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Bispecific and Trispecific Engagers: NK-T Cells and Cancer Therapy
Posted in Cancer and Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy,Immuno-Oncology &
Genomics, immunology, immunotherapy, tagged adoptive cell therapy, allogeneic, cancer, Cancer
immunotherapy, Clinical trial, expansion,immunotherapy, Natural killer cell, natural killer cells, non-HLA-related
donor on May 8, 2016 | Leave a Comment

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Bispecific and Trispecific Engagers: NK-T Cells and Cancer Therapy

Curator: Larry H. Bernstein, MD, FCAP

Successful adoptive transfer and in vivo expansion of human haploidentical NK cells

in patients with cancer
Jeffrey S. Miller, Yvette Soignier, Angela Panoskaltsis-Mortari, , Todd E. Defor, Linda J. Burns, Paul J.
Orchard, Bruce R. Blazar, John E. Wagner, Arne Slungaard, Daniel J. Weisdorf, Ian J. Okazaki, and
Philip B. McGlave
Blood. 2005;105:3051-3057

http://www.fortressbiotech.com/pdfs/Miller_NK%20adoptive

%20immunotherapy.Blood.2005.pdf

We previously demonstrated that autologous natural killer (NK)cell therapy after hematopoietic cell
transplantation (HCT) is safe but does not provide an antitumor effect. We hypothesize that this is due
to a lack of NK-cell inhibitory receptor mismatching with autologous tumor cells, which may be
overcome by allogeneic NK-cell infusions. Here, we test haploidentical, related-donor NK-cell infusions
in a nontransplantation setting to determine safety and in vivo NK-cell expansion. Two lower intensity
outpatient immune suppressive regimens were tested: (1) lowdose cyclophosphamide and
methylprednisolone and (2) fludarabine. A higher intensity inpatient regimen of high-dose
cyclophosphamide and fludarabine (HiCy/Flu) was tested in patients with poorprognosis acute myeloid
leukemia (AML). All patients received subcutaneous interleukin 2 (IL-2) after infusions. Patients who
received lower intensity regimens showed transient persistence but no in vivo expansion of donor
cells. In contrast, infusions after the more intense Hi-Cy/Flu resulted in a marked rise in endogenous
IL-15, expansion of donor NK cells, and induction of complete hematologic remission in 5 of 19 poorprognosis patients with AML. These findings suggest that haploidentical NK cells can persist and
expand in vivo and may have a role in the treatment of selected malignancies used alone or as an
adjunct to HCT.
Human natural killer (NK) cells are a subset of peripheral blood lymphocytes defined by the expression
of CD56 or CD16 and the absence of the T-cell receptor (CD3).1 They recognize and kill transformed
cell lines in a major histocompatibility complex (MHC)unrestricted fashion and produce cytokines
critical to the innate immune response. NK-cell function, distinct from the MHC-restricted cytolytic
activity of T cells, may play a role in antitumor surveillance.2 The effects of NK-cell infusions have
been studied in adoptive immunotherapy clinical trials. In these studies, autologous lymphokineactivated killer cells obtained from peripheral blood mononuclear cells (PBMCs) were administered to
patients along with exogenous high-dose interleukin-2 (IL-2). Up to 20% of patients responded to
these infusions of NK-cell containing populations.3
In contrast to NK cells, T cells recognize targets through an antigen-specific T-cell receptor (TCR) and
interact with targets only if human leukocyte antigen (HLA) MHC antigens are also recognized.
Although NK-cell killing is MHC-unrestricted, NK cells display a number of activating and inhibitory
receptors that ligate MHC molecules to modulate the immune response.4,5 NK-cell receptors that
recognize antigens at the HLA-A, -B, or -C loci are members of the immunoglobulin superfamily and
are termed killer immunoglobulin receptors (KIRs).6,7 Other receptor families (natural killer group 2
[NKG2]/CD94) that recognize antigens of the nonclassical HLA-E, -F, or -G loci and other ligand
specificities have also been described.8-10 Engagement of these NK-cell receptors results in

stimulation or inhibition of NK-cell effector function depending on intracellular signaling mediated

through the cytoplasmic tail or adaptor molecules associated with each receptor.11-13 The NK-cell
response to a target thus depends on the net effect of activating and inhibitory receptors.
Clinical trials have assessed the effects of low-dose IL-2 administration on activation of NK cells in
patients with cancer. We have demonstrated the safety and feasibility of daily subcutaneous IL-2
injections following high-dose chemotherapy and autologous hematopoietic cell transplantation (HCT).
Whereas IL-2 signifi- cantly expanded the number of circulating NK cells in vivo, these NK cells were
not maximally cytotoxic as determined by in vitro assays.14 Subsequent studies tested infusion of IL2activated NK-cellenriched populations or intravenous IL-2 infusions combined with subcutaneous
IL-2. Although these approaches augmented in vivo NK-cell function, no consistent efficacy of
autologous NK-cell therapy could be detected in cancer patients when compared with cohorts of
matched controls.15
We then hypothesized that autologous NK cells may be suppressed by the physiologic response
resulting from NK-cell recognition of self MHC molecules. This notion is supported by recent data
from haploidentical T-celldepleted transplantation studies. KIR mismatch with tumor MHC (ie, KIR
ligand) may lead to greater tumor kill. In these studies, Ruggeri et al16 showed that stratifying
patients by their KIR ligand mismatch would select for patients with alloreactive NK cells that protect
against acute myeloid leukemia (AML) relapse. Although virtually untested in solid tumors, these
clinical data strongly support a therapeutic role for allogeneic NK cells in myeloid leukemia.17 We
present data on the biologic effects of haploidentical NK-cell infusions administered to cancer patients
as cell-based immunotherapy with the goal of demonstrating a feasible and safe method that permits
in vivo donor NK-cell expansion.

In this study, we demonstrate that adoptively transferred human NK cells derived from haploidentical
related donors can be expanded in vivo. Of interest, in vivo NK-cell expansion occurs after preparation
with a high dose (Hi-Cy/Flu) but not lower doses of immunosuppression (Lo-Cy/mPred or Flu).
Successful lymphocyte adoptive transfer following intensive immunosuppresion is not surprising.
Lymphopenia may change the competitive balance between transferred lymphocytes and endogenous
lymphocytes. Alternatively, lymphopenia may induce survival factors or deplete cellular or soluble
inhibitory factors.25,26 In murine studies, preparative regimens sufficient to induce lymphopenia

allowed homeostatic T-cell expansion in vivo that potentiated effective antitumor immunity.27 This
concept has been tested in human T-cell clinical trials by Rosenbergs group.28 T-cell lymphopenia was
induced by Hi-Cy/Flu, similar to what was used here. Successful adoptive transfer and expansion of NK
cells may also require intense immunosuppression. Prlic et al20 showed that mature NK cells
proliferated only in an NK-celldeficient host where the endogenous NK-cell pool was absent.
We also demonstrate that NK-cell adoptive therapy is associated with a striking rise in endogenous IL15 levels, reminiscent of the role IL-7 plays in CD4 T-cell homeostasis.29 IL-15 is required for the final
steps of in vitro NK-cell differentiation from CD34 progenitors.22-24 Cooper et al21 was the first to
show that IL-15 was absolutely required for in vivo expansion and survival of NK cells, in mice, in part
through bcl-2 expression. Transfer of NK cells into IL-15/ hosts resulted in loss of NK cells by 4 days
after transfer. IL-15 receptor alpha knockout mice generate IL-15 but do not have NK cells and are
unable to undergo successful adoptive transfer. This implies that IL-15 responsiveness by cells other
than NK cells may be important in driving this response. IL-15 transgenic mice markedly expand their
NK cells and CD8 T cells, ultimately resulting in an NK/T-lymphocytic leukemia.30 The endogenous
origin of IL-15 in our patients was unclear. Our data support the notion that IL-15 levels increased only
after an intensive lymphocyte-depleting preparative regimen as demonstrated by the inverse
correlation between IL-15 concentrations and the absolute lymphocyte count. This does not exclude
the possibility that IL-15 may be produced following chemotherapy-induced damage to gastrointestinal
mucosa or other cells of epithelial origin.31-34 The effects of exogenous IL-2 administration in these
patients needs to be explored as it does add toxicity to the regimen. Further clinical testing may
demonstrate that expansion will occur in the presence of IL-15 alone.
Donor NK-cell infusions were feasible and tolerated without unexpected toxicity except for the
umbilical cord blood transplantation patient who developed EBV reactivation after treatment. The risk
of posttransplantation lymphoproliferative disease approached 10% when HCT is performed using a Tcelldepleted and mismatched graft.35 Although a single event, this finding is important to understand
the possible consequences of allogeneic NK-cell therapy in heavily pretreated immunosuppressed
patients. It also emphasizes that the CD3- depleted final product, enriched for NK cells but containing
B cells, may need further purification to lessen the possibility of this complication. Clinical ex vivo
selection methods to address this issue using CD3 depletion followed by CD56 selection are now in
place36 and will be tested. We have previously shown that monocytes serve as accessory cells for NKcell expansion in vitro18 but the role of accessory cells in vivo, if any, is unknown. We need to verify

that removal of monocytes and B cells does not change the in vivo expansion potential of NK cells
seen here before recommending a purified NK-cell product in all future studies.
In summary, this is the first study to demonstrate that adoptively transferred human NK cells can be
expanded in vivo. Expansion was dependent on the more intense Hi-Cy/Flu preparative regimen,
which induced lymphopenia, and the more potent immunosuppression that was associated with high
endogenous concentrations of IL-15, none of which was observed following Lo-Cy/mPred and Flu
alone. It is intriguing that this same regimen is the basis for many transplantation regimens and may
help explain the robust NK-cell reconstitution seen in that setting. In this study, NKenriched cells were
obtained from related haploidentical donors by efficient depletion of CD3 from PBMCs, although
contaminating B cells and monocytes remained in the final product. A maximum tolerated dose was
not reached and the largest cell dose administered was that obtained during a single lymphapheresis
collection. Although tumor response was not a primary goal of this study, 5 of 19 poor-prognosis
patients with AML achieved complete remission after haploidentical NK-cell therapy, with a significantly
higher complete remission rate when KIR ligand mismatched donors were used, a strategy that
predicts NK-cell alloreactivity.16,37 The precise role of the cells versus the high-intensity
chemotherapy regimen in responding patients cannot be definitively determined in this current study.
However, the benefit of alloreactivity and the preferential expansion of functional NK cells in
responding patients is consistent with at least a partial effect from the NK cells. Our data suggest that
prospective selection of KIR ligand mismatched donors is warranted when possible, which will be
assessed in subsequent larger clinical trails.

The biology of natural killer cells in cancer, infection, and pregnancy.

Miller JS1

Exp Hematol. 2001 Oct;29(10):1157-68.

http://www.ncbi.nlm.nih.gov/pubmed/11602317

OBJECTIVE: NK cells are important cells of the immune system. They are ultimately derived from
pluripotent hematopoietic stem cells. NK cell cytotoxicity and other functions are tightly regulated by
numerous activating and inhibitory receptors including newly discovered receptors that selectively
recognize major histocompatibility complex class I alleles. Based on their defining function of
spontaneous cytotoxicity without prior immunization, NK cells have been thought to play a critical role
in immune surveillance and cancer therapy. However, new insights into NK cell biology have suggested
major roles for NK cells in infection control and uterine function. The purpose of this review is to
provide an update on NK cell function, ontogeny, and biology in order to better understand the role of
NK cells in health and disease.
DATA SOURCES: In the Medline database, the major subject heading Natural Killer Cells was
introduced in 1983, identifying 16,848 citations as of December 31, 2000. Since 1986, there have
been approximately 1000 citations per year under this subject heading. In this database, 68% of

manuscripts are limited to human NK cells; 40% of citations cross with the major sub-heading of
cytotoxicity, 40% with cytokines, 36% with neoplasm, 5% with antibody-dependent cellular
cytotoxicity, 2.8% with pregnancy, and 1.3% with infection. Of references from the year 2000-2001,
46 were selected to combine with contributions from earlier literature.
CONCLUSIONS: NK cells should no longer be thought of as direct cytotoxic killers alone as they clearly
serve a critical role in cytokine production which may be important to control cancer, infection, and
fetal implantation. Understanding mechanisms of NK cell functions may lead to novel therapeutic
strategies for the treatment of human disease.

NK cell-based immunotherapy for malignant diseases

Min Cheng, Yongyan Chen, Weihua Xiao, Rui Sun and Zhigang Tian
Cellular & Molecular Immunology (2013) 10, 230252;

published online 22 April 2013

http://dx.

doi.org:/10.1038/cmi.2013.10
Natural killer (NK) cells play critical roles in host immunity against cancer. In response, cancers
develop mechanisms to escape NK cell attack or induce defective NK cells. Current NK cell-based
cancer immunotherapy aims to overcome NK cell paralysis using several approaches. One approach
uses expanded allogeneic NK cells, which are not inhibited by self histocompatibility antigens like
autologous NK cells, for adoptive cellular immunotherapy. Another adoptive transfer approach uses
stable allogeneic NK cell lines, which is more practical for quality control and large-scale production. A
third approach is genetic modification of fresh NK cells or NK cell lines to highly express cytokines, Fc
receptors and/or chimeric tumor-antigen receptors. Therapeutic NK cells can be derived from various
sources, including peripheral or cord blood cells, stem cells or even induced pluripotent stem cells
(iPSCs), and a variety of stimulators can be used for large-scale production in laboratories or good
manufacturing practice (GMP) facilities, including soluble growth factors, immobilized molecules or
antibodies, and other cellular activators. A list of NK cell therapies to treat several types of cancer in
clinical trials is reviewed here. Several different approaches to NK-based immunotherapy, such as
tissue-specific NK cells, killer receptor-oriented NK cells and chemically treated NK cells, are
discussed. A few new techniques or strategies to monitor NK cell therapy by non-invasive imaging,
predetermine the efficiency of NK cell therapy byin vivoexperiments and evaluate NK cell therapy
approaches in clinical trials are also introduced.
Surgery, chemotherapeutic agents and ionizing radiation have been used for decades as primary
strategies to eliminate the tumors in patients; however, the development of resistance to drugs or
radiation led to a significant incidence of tumor relapse. Therefore, investigating effective strategies to
eliminate these resistant tumor cells is urgently needed. The importance of immune system in
malignant diseases has been demonstrated by recent major scientific advances.

Both innate and adaptive immune cells actively prevent neoplastic development in a process called
cancer immunosurveillance. Innate immune cells, including monocytes, macrophages, dendritic cells
(DCs) and natural killer (NK) cells, mediate immediate, short-lived responses by releasing cytokines
that directly lyse tumor cells or capture debris from dead tumor cells. Adaptive immune cells, including
T and B cells, mediate long-lived, antigen-specific responses and effective memory.1Despite these
immune responses, malignant cells can develop mechanisms to evade immunosurveillance. Some
tumors protect themselves by establishing an immune-privileged environment. For example, they can
produce immunosuppressive cytokines IL-10 and transforming growth factor- (TGF-) to suppress
the adaptive antitumor immune response, or skew the immune response toward a Th2 response with
significantly less antitumor capacity.2,3,4 Some tumors alter their expressions of IL-6, IL-10, vascular
epithelial growth factor or granulocyte monocyte-colony stimulating factor (GM-CSF), impairing DC
functionsvia inactivation or suppressing maturation.5 In some cases, induced regulatory T cells
suppress tumor-specific CD4+ and CD8+ T-cell responses.6 Tumor cells also minimally express or shed
tumor-associated antigens, shed the ligands of NK cell-activating receptor such as the NKG2D ligands
UL16-binding protein 2, major histocompatibility complex (MHC) class I chain-related molecules A and
B molecules (MICA/MICB) or alter MHC-I and costimulatory molecule expression to evade the immune
responses.7,8,9 Malignant cells may also actively eliminate immune cells by activation-induced cell
death or Fas ligand (FasL) expression.10,11 In addition, primary cancer treatments like chemotherapy
and ionizing radiation can compromise antitumor immune responses by their immunosuppressive side
effects.
Tumor cells can be eliminated when immune responses are adequate; when they are not, tumor
growth and immunourveillance enter into a dynamic balance until tumor cells evade
immunosurveillance, at which point neoplasms appear clinically as a consequence. Therapies designed
to induce either a potent passive or active antitumor response against malignancies by harnessing the
power of the immune system, known as tumor immunotherapy, is an appealing alternative strategy to
control tumor growth. Until now, the cancer immunotherapy field has covered a vast array of
therapeutic agents, including cytokines, monoclonal antibodies, vaccines, adoptive cell transfers (T, NK
and NKT) and Toll-like receptor (TLR) agonists.1,12,13 Adoptive NK cell transfer in particular has held
great promise for over three decades. With progress in the NK cell biology field and in understanding
NK function, developing NK cells to be a powerful cancer immunotherapy tool has been achieved in
recent years. In this article, we will review recent advances in NK cell-based cancer immunotherapy,
focusing on potential approaches and large-scale NK cell expansion for clinical practice, as well as on
the clinical trials and future perspectives to enhance the efficacy of NK cells.

NK cells were first identified in 1975 as a unique lymphocyte subset that are larger in size than T and
B lymphocytes and contain distinctive cytoplasmic granules. 14,15After more than 30 years, our
understanding of NK cell biology and function lends important insights into their role in
immunosurveillance. It has been known that NK cells develop in bone marrow (BM) from common
lymphoid progenitor cells;16however, NK cell precursors have still not been clearly characterized in
humans.17After development, NK cells distribute widely throughout lymphoid and non-lymphoid
tissues, including BM, lymph nodes (LN), spleen, peripheral blood, lung and liver.18
NK cells, defined as CD3CD56+ lymphocytes, are distinguished as CD56bright and CD56dim subsets.
Approximately 90% of peripheral blood and spleen NK cells belong to the CD56 dimCD16+ subset with
marked cytotoxic function upon interacting with target cells. 19,20In contrast, most NK cells in lymph
nodes and tonsils belong to the CD56brightCD16 subset and exhibit predominantly immune regulation
properties by producing cytokines such as interferon (IFN)- in response to IL-12, IL-15 and IL-18
stimulation.19,21
NK cells rapidly kill certain target cells without prior immunization or MHC restriction, whose activation
is dependent on the balance between inhibitory and activating signals from invariant
receptors.22,23,24 The activating receptors include the cytotoxicity receptors (NCRs) (NKp46, NKp30 and
NKp44), C-type lectin receptors (CD94/NKG2C, NKG2D, NKG2E/H and NKG2F) and killer cell
immunoglobulin-like receptors (KIRs) (KIR-2DS and KIR-3DS), while the inhibitory receptors include
C-type lectin receptors (CD94/NKG2A/B) and KIRs (KIR-2DL and KIR-3DL). Since some structural
families contain both activating and inhibitory receptors, trying to understand how NK cell activity is
regulated is often complicated.25 At steady state, the inhibitory receptors (KIRs and CD94/NKG2A/B),
which bind to various MHC-I molecules present on almost all cell types, inhibit NK cell activation and
prevent NK cell-mediated killing. Under stress conditions, cells downregulate MHC-I expression,
causing NK cells to lose inhibitory signaling and be activated in a process called missing-self
recognition. Additionally, the non-MHC self molecules Clr-b (mouse), LLT-1 (human) and CD48
(mouse) recognized by the inhibitory receptors NKR-P1B, NKR-P1A and 2B4, respectively, also perform
this function.26,27 In contrast to the self-expressed inhibitory receptor ligands, NK cell-activating
receptors can recognize either pathogen-encoded molecules that are not expressed by the host, called
non-self recognition, or self-expressed proteins that are upregulated by transformed or infected cells,
called stress-induced self recognition. For example, mouse Ly49H recognizes cytomegalovirusencoded m157, and NKG2D recognizes the self proteins human UL16-binding proteins and
MICA/MICB.28,29 NK cells identify their targets by recognizing a set of receptors on target cells in an
NK-target cell zipper formation; this results in the integration of multiple activating and inhibitory

signals, the outcome of which depends on the nature of the interacting cells. 26IFNs or DC/macrophagederived cytokines, such as type I IFN, IL-12, IL-18 and IL-15, enhance the activation or promote the
maturation of NK cells, which can also augment NK cell cytolytic activity against tumor
cells.30,31,32 Cytotoxic activity of NK cells can increase approximately 20200 fold after exposure to IFN/ or IL-12. Despite these known innate immune cell functions, accumulating evidence in both mice
and humans demonstrates that NK cells are educated and selected during development, possess
receptors with antigen specificity, undergo clonal expansion during infection and can generate longlived memory cells.33,34
After over 30 years of researching NK cells, evidence supports that they play critical roles in the early
control of viral infection, in hematopoietic stem cell (HSC) transplantation (improved grafting, graftvs.-host disease and graft-vs.-tumor), in tumor immunosurveillance and in reproduction (uterine spiral
artery remodeling). The roles of NK cells in controlling organ transplantation, parasitic and HIV
infections, autoimmunity and asthma have also been suggested, but remain to be explored
further.26 In particular, therapeutic strategies harnessing the power of NK cells to target multiple
malignancies have been designed.
NK cells originally described as large granular lymphocytes, exhibited natural cytotoxicity against
certain tumor cells in the absence of preimmunization or stimulation.35,36,37 CD56dim NK cells,
which make up the majority of circulating cells, are the most potent cytotoxic NK cells against tumor
cells. Evidence gathered from a mouse xenograft tumor model testing functionally deficient NK cells or
antibody-mediated NK cell depletion supports that NK cells can eradicate tumor cells.38,39,40,41 An
11-year follow-up study in patients indicated that low NK-like cytotoxicity was associated with
increased cancer risk.42 High levels of tumor infiltrating NK cells (TINKs) are associated with a
favorable tumor outcome in patients with colorectal carcinoma, gastric carcinoma and squamous cell
lung cancer, suggesting that NK-cell infiltration into tumor tissues represents a positive prognostic
marker.43,44,45 As described above, NK-cell recognition of tumor cells by inhibitory and activating
receptors is complex, and the three recognition modelsmissing-self, non-self and stress-induced
selfmight be used to sense missing- or altered-self cells. Activated NK cells are thus in a position to
directly or indirectly exert their antitumor activity to control tumor growth and prevent the rapid
dissemination of metastatic tumors by immunosurveillance mechanisms (Figure 1).

Figure 1.
NK cells in tumor immunosurveillance. The diagram shows the potential roles of NK cells in tumor
immunosurveillance. NK cells initially recognize the tumor cells viastress or danger signals. Activated
NK cells directly kill target tumor cells through at least four mechanisms: cytoplasmic granule release,
death receptor-induced apoptosis, effector molecule production or ADCC. Additionally, NK cells act as
regulatory cells when reciprocally interact with DCs to improve their antigen uptake and presentation,
facilitating the generation of antigen-specific CTL responses. Also, by producing cytokines such as IFN, activated NK cells induce CD8+ T cells to become CTLs. Activated NK cells can also promote
differentiation of CD4+ T cells toward a Th1 response and promote CTL differentiation. Cytokines
produced by NK cells might also regulate antitumor Ab production by B cells. Ab, antibody; ADCC,

antibody-dependent cellular cytotoxicity; CTL, cytotoxic T lymphocyte; DC, dendritic cell; IFN,
interferon; NK, natural killer.
Full figure and legend (96K)

Direct tumor clearance by NK-mediated cytotoxicity

Upon cellular transformation, surface MHC-I expression on tumor cells is often reduced or lost to
evade recognition by antitumor T cells. In parallel, cellular stress and DNA damage lead to upregulated
expression of ligands on tumor cells for NK cell-activating receptors. Human tumor cells that have lost
self MHC-I expression or bear altered-self stress-inducible proteins are ideal NK cell targets, as NK
cells are activated by initially recognizing certain stress or danger signals. 46 The missing-self model
of tumor cell recognition by NK cells was first demonstrated by observing that MHC-I-deficient
syngeneic tumor cells were selectively rejected by NK cells; additionally, NK cell inhibitory receptors
were shown to detect this absence of MHC-I expression. 47,48,49 NK cells can also kill certain MHC-Isufficient tumor cells by detecting stress-induced self ligands through their activating receptors. Broad
MICA/B expression has been detected on epithelial tumors, melanoma, hepatic carcinoma and some
hematopoetic malignancies, representing a counter-measure by the immune system to combat tumor
development.31 NK cell-mediated cytotoxicity is also important against tumor initiation and
metastasis in vivo.50,51,52
NK cells directly kill target tumor cells through several mechanisms: (i) by releasing cytoplasmic
granules containing perforin and granzymes that leads to tumor-cell apoptosis by caspase-dependent
and -independent pathways.53,54 Cytotoxic granules reorient towards the tumor cell soon after NK
tumor cell interaction and are released into the intercellular space in a calcium-dependent manner;
granzymes are allowed entry into tumor cells by perforin-induced membrane perforations, leading to
apoptosis; (ii) by death receptor-mediated apoptosis. Some NK cells express tumor-necrosis factor
(TNF) family members, such as FasL or TNF-related apoptosis-inducing ligand (TRAIL), which can
induce tumor-cell apoptosis by interacting with their respective receptors, Fas and TRAIL receptor
(TRAILR), on tumor cells. 55,56,57,58,59 TNF- produced by activated NK cells can also induce tumor-cell
apoptosis;60 (iii) by secreting various effector molecules, such as IFN-, that exert antitumor functions
in various ways, including restricting tumor angiogenesis and stimulating adaptive
immunity.61,62 Cytokine activation or exposure to tumor cells is also associated with nitric oxide (NO)
production, where NK cells kill target tumor cells by NO signaling; 63,64 (iv) through antibody-dependent
cellular cytotoxicity (ADCC) by expressing CD16 to destroy tumor cells. 40 The antitumor activity of NK
cells can be further enhanced by cytokine stimulation, such as by IL-2, IL-12, IL-18, IL-15 or those
that induce IFN production.40,65,66,67,68,69,70

Indirect NK-mediated antitumor immunity

NK cells act as regulatory cells when reciprocally interact with DCs, macrophages, T cells and
endothelial cells by producing various cytokines (IFN-, TNF- and IL-10), as well as chemokines and
growth factors.26,71 By producing IFN-, activated NK cells induce CD8 + T cells to become cytotoxic T
lymphocytes (CTLs), and also help to differentiate CD4+ T cells toward a Th1 response to promote CTL
differentiation.72,73 NK cell-derived cytokines might also regulate antitumor antibody (Ab) production
by B cells.40 In addition, cancer cells killed by NK cells could provide tumor antigens for DCs, inducing
them to mature and present antigen.74By lysing surrounding DCs that have phagocytosed and
processed foreign antigens, activated NK cells also could provide additional antigenic cellular debris for
other DCs. Thus, activated NK cells promote antitumor immunity by regulating DC activation and
maturation,75 as these DCs can facilitate the generation of antigen-specific CTL responses through
their ability to cross-present tumor-specific antigens (derived from NK cell-mediated tumor lysis) to
CD8+ T cells.76,77
During tumor progression, tumor cells develop several mechanisms to either escape from NK-cell
recognition and attack or to induce defective NK cells. These include losing expression of adhesion
molecules, costimulatory ligands or ligands for activating receptors, upregulating MHC class I, soluble
MIC, FasL or NO expression, secreting immunosuppressive factors such as IL-10, TGF- and indoleam
ine 2,3-d ioxygense (IDO) and resisting Fas- or perforin-mediated apoptosis. 31,78,79 In cancer patients,
NK cell abnormalities have been observed, including decreased cytotoxicity, defective expression of
activating receptors or intracellular signaling molecules, overexpression of inhibitory receptors,
defective proliferation, decreased numbers in peripheral blood and in tumor infiltrate, and defective
cytokine production.60Given that NK cells play critical roles in the first-line of defense against
malignancies by direct and indirect mechanisms, the therapeutic use of NK cells in human cancer
immunotherapy has been proposed and followed in a clinical context (Table 1).
Table 1 NK cells in tumor immunotherapy.

Full table

.more
For NK cell immunotherapy, obtaining a sufficient number of functional NK cells is critical in clinical
protocols. Therefore, the number, purity and state of NK cell proliferation and activation are considered
as the key factors.151 In Table 2, the purification/expansion of clinical-grade NK cells developed in
recent years is summarized. They can be produced from cord blood, bone marrow, peripheral blood
and embryonic stem cells. Overall, the summarized methods suggest that long-term ex vivoexpansion
of NK cells may present a clinical benefit, but not the short-term activation which is not sufficient for
augmenting the functions of NK cells.152

Table 2 Expansion of NK cells in vitro for clinical practice*.Full table

.more
Results from treating hematological malignancies demonstrated a critical role for NK cells in clinical
immunotherapy, as alloreactive NK cells highlighted the graft-vs.-leukemia effect in AML
patients.172 The graft-vs.-tumor effect of alloreactive NK cells was also strengthened by mismatched
IL-2-activated lymphocytes in patients with solid tumors or hematological malignancies. 173 As
discussed above, autologous NK cells, allogeneic NK cells, NK cell lines and genetically modified NK
cells were investigated for effectiveness as tumor immunotherapies. The clinical study designs
evaluating the efficacy of these various NK cell-mediated tumor therapies are summarized in Table 3.
Table 3 Clinical trials of tumor immunotherapy by using NK cells. Full table
..more
NK cell-based immunotherapy holds great promise for cancer treatment. However, only modest clinical
success has been achieved thus far using NK cell-based therapies in cancer patients. Progress in the
field of understanding NK cell biology and function is therefore needed to assist in developing novel
approaches to effectively manipulate NK cells for the ultimate benefit of treating cancer patients.

Present and Future of Allogeneic Natural Killer Cell Therapy

Okjae Lim,1 Mi Young Jung,1 Yu Kyeong Hwang,2 and Eui-Cheol Shin3,*
Front Immunol. 2015; 6: 286. Published online 2015 Jun 3.

doi: 10.3389/fimmu.2015.00286

Natural killer (NK) cells are innate lymphocytes that are capable of eliminating tumor cells
and are therefore used for cancer therapy. Although many early investigators used
autologous NK cells, including lymphokine-activated killer cells, the clinical efficacies were
not satisfactory. Meanwhile, human leukocyte antigen (HLA)-haploidentical hematopoietic
stem cell transplantation revealed the antitumor effect of allogeneic NK cells, and HLAhaploidentical, killer cell immunoglobulin-like receptor ligand-mismatched allogeneic NK
cells are currently used for many protocols requiring NK cells. Moreover, allogeneic NK cells
from non-HLA-related healthy donors have been recently used in cancer therapy. The use of
allogeneic NK cells from non-HLA-related healthy donors allows the selection of donor NK
cells with higher flexibility and to prepare expanded, cryopreserved NK cells for instant
administration without delay for ex vivo expansion. In cancer therapy with allogeneic NK
cells, optimal matching of donors and recipients is important to maximize the efficacy of the

therapy. In this review, we summarize the present state of allogeneic NK cell therapy and
its future directions.
Cancer is a major threat for humans worldwide, with approximately 14 million new cases and 8.2
million cancer-related deaths in 2012 (1). Although most common cancer treatments include surgery,
chemotherapy, and radiotherapy, unsatisfactory cure rates require new therapeutic approaches,
especially for refractory cancers. For this purpose, cancer immunotherapies with various cytokines,
antibodies, and immune cells have been clinically applied to patients to encourage their own immune
system to help fight the cancer (2).
Adoptive cellular immunotherapies have employed several types of immune cells, including dendritic
cells (DCs), cytotoxic T lymphocytes (CTLs), lymphokine-activated killer (LAK) cells, cytokine-induced
killer (CIK) cells, and natural killer (NK) cells. Although there has been recent progress in DC therapy
and CTL therapy, clinical applications are somewhat limited because cancer antigens must first be
characterized and autologous cells must be used. By contrast, LAK cells, CIK cells, and NK cells have
antigen-independent cytolytic activity against tumor cells. In particular, NK cells can be used from not
only autologous sources but also allogeneic sources and, recently, allogeneic NK cells have been
employed more often in cancer treatment. Whereas autologous NK cells from cancer patients may
have functional defects (3), allogeneic NK cells from healthy donors have normal function and can be
safely administered to cancer patients (4). Allogeneic NK cell therapy is particularly beneficial because
it can enhance the anti-cancer efficacy of NK cells via donorrecipient incompatibility in terms of killer
cell immunoglobulin-like receptors (KIRs) on donor NK cells and major histocompatibility complex
(MHC) class I on recipient tissues.
Natural killer cells are innate lymphocytes that provide a first line of defense against viral infections
and cancer (5). Human NK cells are recognized as CD3CD56+lymphocytes. They can be further
subdivided into two subsets based on the surface expression level of CD56. The CD56 dim population
with low-density expression of CD56 comprises approximately 90% of human blood NK cells and has a
potent cytotoxic function, whereas the CD56bright population (approximately 10% of blood NK cells)
with high-density expression of CD56 displays a potent cytokine producing capacity and has
immunoregulatory functions (6). The CD56dim NK cell subset also expresses high levels of the Fc
receptor for IgG (FcRIII, CD16), which allows them to mediate antibody-dependent cellular
cytotoxicity (ADCC) (7). NK cells comprise 515% of circulating lymphocytes and are also found in
peripheral tissues, including the liver, peritoneal cavity, and placenta. Activated NK cells are capable of
extravasation and infiltration into tissues that contain pathogens or malignant cells while resting NK
cells circulate in the blood (8).

The NK cell activity is regulated by signals from activating and inhibitory receptors (9, 10). The
activating signal is mediated by several NK receptors including NKG2D and natural cytotoxicity
receptors (NCRs) (911). By contrast, NK cell activity is suppressed by inhibitory receptors, including
KIRs, which bind to human leukocyte antigen (HLA) class I molecules on target cells (9, 10, 12).
NKG2A is also an important inhibitory receptor binding to non-classical HLA molecule, HLA-E (13). If
target cells lose or downregulate HLA expression (14), the NK inhibitory signal is abrogated, allowing
NK cells to become activated and kill malignant targets. However, NK cell function is impaired in
cancer patients by various mechanisms, particularly in tumor microenvironment (15).
Although NK cell activity is determined by the summation of signals from activating and inhibitory
receptors, the inhibitory signal through KIRs is a main regulator of NK cell function particularly in
allogeneic settings. Inhibitory KIRs have long cytoplasmic tails containing two immunoreceptor
tyrosine-based inhibition motifs (ITIMs). Each KIR has its cognate ligand and consists of two (KIR2DL)
or three (KIR3DL) extracellular Ig-domains. KIR2DL1 and KIR2DL2/3 recognize group 2 HLA-C (called
C2, Lys80) and group 1 HLA-C (called C1, Asn80), respectively. KIR3DL1 recognizes HLA-Bw4 (16).
The KIR repertoire on human NK cells is randomly determined and independent of the number and
allotype of HLA class I ligands (17).
The antitumor activity of allogeneic NK cells has been demonstrated in the setting of hematopoietic
stem cell transplantation (HSCT). Allogeneic HSCT is an established curative treatment for hematologic
malignancies. In allogeneic HSCT, donor T cells contribute to graft-versus-host disease (GVHD) and
graft-versus-tumor (GVT) effects (18). In T cell-depleted HSCT, however, donor NK cells are the major
effector cells responsible for controlling residual cancer cells before T cell reconstitution (19, 20).
Natural killer cells are the first lymphoid population to recover after allogeneic HSCT. In the first month
of transplantation, reconstituted NK cells represent the predominant lymphoid cells and play a crucial
role in controlling the host immune system. Allogeneic NK cells prevent viral infections and restrain
residual cancer cells in the early phase of transplantation (21). Of note, the GVT activity of donor NK
cells is significantly improved when KIRs of donor and HLA class I of the recipient are incompatible,
and consequently when inhibitory signals are absent, as observed in HLA-haploidentical HSCT (22).
Therefore, increased GVT activity of NK cells with KIR-HLA incompatibility is the underlying rationale
for the development of allogeneic NK cell therapy.
Following the discovery of inhibitory KIRs and the understanding that they play a role in preventing NK
cell killing of self MHC class I-expressing tumor cells, investigators began to research the possibility of
using allogeneic donor NK cells instead of autologous NK cells for cancer therapy. Several groups have
infused activated, expanded donor NK cells to patients early after allogeneic HSCT to provide

antitumor effects (23). In Table Table1,1, clinical trials with allogeneic NK cells as therapeutics are
summarized.
Table 1

Selected clinical trials with expanded allogeneic NK cells

.. more
As summarized in Table Table2,2, two clinical trials are investigating the use of CAR-expressing
allogeneic NK cells. The aim of both studies is to assess the safety, feasibility, and efficacy of
expanded, activated, and CD19-redirected haploidentical NK cells in ALL patients who have persistent
disease after intensive chemotherapy or HSCT (NCT00995137, NCT01974479). Further, other tumor
antigens, such as CS1, CEA, CD138, and CD33, are targeted by CARs expressed by NK cells, although
NK-92, YT, or NKL cell lines were used (4851).
Table 2 Genetically modified, expanded allogeneic NK cells.
.

Therapeutic regimens
In allogeneic NK cell therapy, optimal therapeutic regimens for clinical applications should be
considered because adoptively transferred NK cells not only target tumor cells but also interact with
the immunological environment. To potentiate the therapeutic efficacy of allogeneic NK cells, proper
strategies, including pre-conditioning or combination therapy, could be applied (34).
Upregulation of NKG2D ligands by spironolactone (63) or histone deacetylase inhibitors (64, 65)
and upregulation of TRAIL-R2 by doxorubicin (66) result in enhanced antitumor efficacy of NK cells.
Proteasome inhibitors also sensitize tumor cells to NK cell-mediated killing via TRAIL and FasL
pathways. In addition, c-kit tyrosine kinase inhibitor (67) and JAK inhibitors (68) increase the
susceptibility of tumor cells to NK cytotoxicity and enhance antitumor responses by increased IFN-
production from NK cells. However, protein kinase inhibitors should be used cautiously because some
protein kinase inhibitors, such as sorafenib, inhibit the effector function of NK cells (69).
Immunomodulatory drugs can augment NK cell function. Lenalidomide enhances rituximab-induced
killing of non-Hodgkins lymphoma and B-cell chronic lymphocytic leukemia through NK cell and
monocyte-mediated ADCC mechanisms (70). Combination therapy using IL-2 and anti-CD25 shows
anti-leukemic effects by depletion of regulatory T cells in addition to activation and expansion of NK
cells (71). Alloferon, an immunomodulatory peptide, enhances the expression of NK-activating
receptor 2B4 and granule exocytosis from NK cells against cancer cells (72).
Therapeutic antibodies can be combined with allogeneic NK cell therapy (73). Antibodies against
tumor antigens (e.g., CD20 and CS1) can induce ADCC of NK cells (74, 75). Antibodies to activating
NK receptors (e.g., 4-1BB, GITR, NKG2D, DNAM-1, and NCRs) can enhance NK activation (74, 7679).
In addition, inhibitory receptors (e.g., KIR2DL, PD-1, PD-L1, and NKG2A) can be blocked by antibodies

(8085). Bispecific and trispecific killer cell engagers directly activate NK cells through CD16 signaling
and thus, induce cytotoxicity and cytokine production against tumor targets (86, 87).
Conclusion
Antitumor activity of allogeneic NK cells was first observed in a setting of HLA-haploidentical HSCT.
Allogeneic NK cell therapy was tried mostly using HLA-haploidentical NK cells with or without
allogeneic HSCT and, recently, allogeneic NK cells from unrelated, random donors have been used in a
non-HSCT setting. The efficacy of allogeneic NK cell therapy can be enhanced by optimal donor
selection in terms of the KIR genotype of donors and donor KIR-recipient MHC incompatibility.
Furthermore, efficacy can be increased by genetic modification of NK cells and optimized therapeutic
regimens. In the future, allogeneic NK cell therapy can be an effective therapeutic modality for cancer.

T cells for immune therapy of patients with lymphoid malignancies

http://dx.doi.org:/10.1182/blood-2002-12-3665

Prepublished online Blood March 6,

2003; 2003 102: 200-206

Martin Wilhelm, Volker Kunzmann, Susanne Eckstein, Peter Reimer, Florian Weissinger,
Thomas Ruediger and Hans-Peter Tony
There is increasing evidence that gammadelta T cells have potent innate antitumor activity. We
described previously that synthetic aminobisphosphonates are potent gammadelta T cell stimulatory
compounds that induce cytokine secretion (ie, interferon gamma [IFN-gamma]) and cell-mediated
cytotoxicity against lymphoma and myeloma cell lines in vitro. To evaluate the antitumor activity of
gammadelta T cells in vivo, we initiated a pilot study of low-dose interleukin 2 (IL-2) in combination
with pamidronate in 19 patients with relapsed/refractory low-grade non-Hodgkin lymphoma (NHL) or
multiple myeloma (MM). The objectives of this trial were to determine toxicity, the most effective dose
for in vivo activation/proliferation of gammadelta T cells, and antilymphoma efficacy of the
combination of pamidronate and IL-2. The first 10 patients (cohort A) who entered the study received
90 mg pamidronate intravenously on day 1 followed by increasing dose levels of continuous 24-hour
intravenous (IV) infusions of IL-2 (0.25 to 3 x 106 IU/m2) from day 3 to day 8. Even at the highest IL2 dose level in vivo, gammadelta T-cell activation/proliferation and response to treatment were
disappointing with only 1 patient achieving stable disease. Therefore, the next 9 patients were
selected by positive in vitro proliferation of gammadelta T cells in response to pamidronate/IL-2 and
received a modified treatment schedule (6-hour bolus IV IL-2 infusions from day 1-6). In this patient
group (cohort B), significant in vivo activation/proliferation of gammadelta T cells was observed in 5
patients (55%), and objective responses (PR) were achieved in 3 patients (33%). Only patients with

significant in vivo proliferation of gammadelta T cells responded to treatment, indicating that

gammadelta T cells might contribute to this antilymphoma effect. Overall, administration of
pamidronate and low-dose IL-2 was well tolerated. In conclusion, this clinical trial demonstrates, for
the first time, that gammadelta T-cell-mediated immunotherapy is feasible and can induce objective
tumor responses.
Despite signicant improvement in the treatment of low-grade non-Hodgkin lymphoma (NHL) and
multiple myeloma (MM), most patients relapse or become resistant to conventional treatment
strategies such as chemotherapy or radiation. Therefore, there is need for alternative tumor therapies.
One possibility is manipulating the immune system to target and eliminate neoplastic cells. Most
current immunotherapeutic approaches aim at inducing antitumor response via stimulation of the
adaptive immune system, which is dependent on major histocompatibility complex (MHC) restricted T
cells. Despite major advances in our understanding of the adaptive immunity toward tumors and the
introduction of vaccine-based strategies, durable responses are rare, and active immunotherapy is still
not an established treatment modality. Adaptive immunotherapeutic approaches have several
disadvantages: T cells need specic tumor-associated antigens (TAAs) and appropriate costimulatory
molecules for activation. Failure or loss of TAAs, MHC molecules, and/or costimulatory molecules
renders tumor cells resistant to T-cellmediated cytotoxicity or induces anergy of specic T cells.1
Mice decient in innate effector cells such as natural killer (NK) cells, NK T cells, or T cells show a
signicantly increased incidence of tumors and provide clear evidence for an immune surveillance
function of the innate immune system.2-4 Recognition of transformed cells by the innate immune
system seems to be dependent on expression of stress-induced ligands and/or loss of MHC class I
molecules on tumor cells.5 Several studies have demonstrated a role for human T cells in recognition
of transformed cells.6,7 T cells exhibit a potent MHC-unrestricted lytic activity against different tumor
cells in vitro.8-10 In addition, T cells have been found with increased frequency in disease-free
survivors of acute leukemia following allogeneic bone marrow transplantation.11 Adoptive transfer of
ex vivoexpanded human T cells in a mouse tumor model further supports the in vivo antitumor
effects of T cells.12 V9V2 T cells, which represent most of the human circulating T cells, recognize
small nonpeptide compounds with an essential phosphate residue (ie, microbial metabolites) or
alkylamines.13-17 As we have shown previously, also synthetic aminobisphosphonates such as
pamidronate are potent T-cell stimulatory compounds.18 In addition, we could demonstrate that
pamidronate-activated T cells produce cytokines (ie, interferon [IFN-]), exhibit specic cytotoxicity
against lymphoma or myeloma cell lines, and lead to reduced survival of autologous myeloma cells.8

The aim of this pilot study is to evaluate the feasibility of activation and/or expansion of T cells in vivo
using the combination of pamidronate and interleukin 2 (IL-2) in patients with refractory/relapsed
lymphoma or myeloma, to determine the most effective IL-2 dose, to assess the toxicity of this
regimen, and to evaluate its ability to exert antitumor effects.

..

There has been no study published so far on in vivo stimulation of T cells in humans, and the
consequences of a selective activation of T cells in vivo were not known. Therefore, evaluation of
toxicity was one major end point of this study. We started with a low IL-2 dose of 0.25 106 IU IL-2/m2
and subsequently increased the IL-2 dose to 3 106 IU IL-2/m2 in cohort A and to 2 106 IU IL-2/m2 in
cohort B. Overall, the combination of pamidronate and IL-2 was well tolerated, and no dose-limiting
toxicity was observed. Most of the patients developed self-limiting fever and thrombophlebitis at the
infusion site. Local thrombophlebitis has been described as a rare side effect in
patients receiving pamidronate alone.20,21 The high frequency of local thrombophlebitis in patients
receiving pamidronate in combination with IL-2 might reect immune-mediated effects on endothelial
cells. It has also been recently shown that aminobisphosphonates have dose-dependent effects on
proliferation-inhibition and apoptosis-induction of human endothelial cells in vitro.22
Next we asked whether the combination of pamidronate and IL-2 induces activation and proliferation
of T cells in vivo. None of the rst 10 patients included in this pilot study (cohort A, Table 1) developed
a measurable T-cell response in vivo. The inability to induce T-cell proliferative response in vivo
correlated with the negative in vitro proliferation of T cells in response to pamidronate/IL-2 in 4 of 5
analyzable patients. Therefore, extensive prior in vitro testing was initiated for all further eligible
patients. Using this strategy, we found that a much lower proportion of patients with hematologic
malignancies showed positive in vitro proliferation of T cells in response to pamidronate/IL-2 compared
with a control group of healthy donors (49% versus 88%). Although the exact mechanisms of this
defect are currently under investigation, a severe immunodeciency caused by extensive prior
chemotherapy in these relapsed/ refractory patients and/or the underlying disease itself may account
for this observation. Indeed, the type of underlying disease seems to inuence the in vitro proliferative
response to pamidronate/IL-2 (Table 2). The failure of patients with B-CLL to develop a measurable Tcell proliferative response may be a result of the very small number of T cells in peripheral blood,
which were often below the detection limit in our series. However, a larger number of patients with
distinct disease entities and at different disease stages (eg, untreated versus treated) need to be
evaluated to support this observation and to identify additional clinical parameters inuencing T-cell
reactivity. Furthermore, extensive prior in vitro testing in eligible patients revealed that T-cell

proliferation in response to pamidronate can be signicantly enhanced by concomitant addition of IL-2

to PBMC cultures on day 1 instead of day 3 (as previously done).
Thus, for all further patients the treatment schedule was changed (concomitant administration of IL-2
on day 1), and only patients with signicant in vitro proliferation of T cells in the presence of
pamidronate and IL-2 were included (cohort B, Table 1). After these modications, signicant in vivo
expansion of T cells could be observed in 5 of 9 patients (55%) (Table 1). In vivo proliferation of T
cells was associated with a robust up-regulation of early (CD69) and late (HLA-DR) activation markers,
whereas pamidronate and IL-2 failed to induce comparable effects on T cells and NK cells (Table 3).
These data support in vitro ndings that the action of pamidronate is highly specic and, except for
V9V2 T cells, it does not activate other immune effector cells.8,23,24 However, at higher IL-2 doses
unspecic stimulation effects of IL-2 became more evident because a proportion of patients showed a
moderate up-regulation of activation markers on T cells and NK cells at the highest dose level of IL-2
tested in this study. On the basis of the analysis of activation marker expression and proliferation we
conclude that 1 106 IU IL-2/m2 IL-2 per day seems to be the most effective dose with respect to
specic and effective T-cell stimulation in vivo.
Another aim of our study was to assess the clinical response. None of the 9 analyzable patients of
cohortA(Table 1) achieved an objective tumor response. After change of protocol and inclusion criteria
(cohort B, Table 1) 3 of 9 patients (33%) achieved an objective tumor response (3 PR). Clinical
response could be associated with T-cell proliferation in vivo, because all 4 patients from cohort B
without T-cell proliferation in vivo did not experience an objective tumor response, and 4 of 5 patients
with T-cell proliferation in vivo responded (3 PR, 1 stable disease [SD]). These results suggest that the
observed tumor regression in our patients is dependent on T-cell activation and proliferation. The
relevance of this correlation is underlined by the fact that pamidronate-stimulated T cells possess an
increased capacity for killing tumor cells in vitro.8,10 It is still open which mechanisms may have been
responsible for the clinical responses. Several other antitumor effects have been attributed to
aminobisphosphonates. However, at pharmacologically achievable concentrations in vivo, only the
specic stimulation of V9V2T cells can be observed.8 Alternatively, the occurrence of clinical
remissions may be attributed to an IL-2mediated effect on other immune effector cells. However, our
immunologic monitoring indicates that the combination of pamidronate and low-dose IL-2 does not
induce specic activation and expansion of T cells or NK cells compared with the effect on T cells. In
addition, the concentrations of IL-2 used here are much lower than the doses required in other
immunotherapeutic approaches for these malignancies.25-27

The important question of what precise mechanisms are involved in tumor recognition and eradication
by T cells is out of the scope of this study and will require further in vitro and in vivo studies. However,
tumor cell recognition by T cells seems to be modulated by a balance of positive and negative
signals.28 Although killer inhibitory receptors (KIRs) are obviously involved in the mediation of
negative signals, the positive signals are only incompletely understood. One example of such a
positive signal is the NKG2D-DAP10 receptor complex, which is known to interact with stress-induced
ligands on tumor cells such as MICA and Rae-1.29 The very slow response proles of most of the
patients in our series strongly argue for an indirect inuence on lymphoma cells rather than a sole
cytotoxic effect. One possible mechanism may be secretion of cytokines, which inuence tumor cells or
their microenvironment.30 We have already shown that IFN- is the major cytokine secreted by
pamidronate-activated T cells.8,31 IFN- has multiple antitumor effects such as direct inhibition of
tumor growth, blocking angiogenesis, or stimulation of macrophages.32 Recently, a signicant
negative correlation between angiogenetic factors (ie, VEGF) and IFN- serum levels was described in
patients treated with pamidronate.33 Therefore, IFN- might be one of the key cytokines involved in
the T-cell mediated antitumor response.
In conclusion, this study indicates for the rst time that in vivo T-cell stimulation by pamidronate and
low-dose IL-2 is a safe and promising immunotherapy approach in the treatment of
patients with low-grade B-NHL and MM. Further studies are necessary to conrm the clinical efcacy of
this novel strategy. Our immunologic and clinical monitoring data provide further insight into the
capacity of T cells to induce an antitumor immune response. However, this study also reveals that the
function of T cells can be impaired in some patients with lymphoid malignancies. Therefore, the results
of this study provide principles relevant to the design of future trials, including appropriate prior in
vitro testing.
EXPANSION OF HIGHLY CYTOTOXIC HUMAN NATURAL KILLER CELLS FOR CANCER CELL
THERAPY
Hiroyuki Fujisaki,1Harumi Kakuda,1Noriko Shimasaki,1Chihaya Imai,1Jing Ma,3Timothy Lockey,4Paul
Eldridge,4Wing H. Leung,1,5 and Dario Campana1,2,5
Cancer Res. 2009 May 1; 69(9): 40104017.

Published online 2009 Apr 21.

doi: 10.1158/0008-5472.CAN-08-3712
Infusions of natural killer (NK) cells are an emerging tool for cancer immunotherapy. The development
of clinically applicable methods to produce large numbers of fully functional NK cells is a critical step to
maximize the potential of this approach. We determined the capacity of the leukemia cell line K562
modified to express a membrane-bound form of interleukin-15 and 4-1BB ligand (K562-mb15-41BBL)
to generate human NK cells with enhanced cytotoxicity. Seven-day coculture with irradiated K562-

mb15-41BBL induced a median 21.6-fold expansion of CD56 +CD3 NK cells from peripheral blood
(range, 5.1-86.6-fold; n = 50), which was considerably superior to that produced by stimulation with
interleukin (IL)-2, IL-12, IL-15 and/or IL-21 and caused no proliferation of CD3 + lymphocytes. Similar
expansions could also be obtained from the peripheral blood of patients with acute leukemia
undergoing therapy (n = 11). Comparisons of the gene expression profiles of the expanded NK cells
and of their unstimulated or IL-2-stimulated counterparts demonstrated marked differences. The
expanded NK cells were significantly more potent than unstimulated or IL-2-stimulated NK cells
against acute myeloid leukemia (AML) cells in vitro. They could be detected for more than one month
when injected into immunodeficient mice and could eradicate leukemia in murine models of AML. We
therefore adapted the K562-mb15-41BBL stimulation method to large-scale clinical-grade conditions,
generating large numbers of highly cytotoxic NK cells. The results that we report here provide
rationale and practical platform for clinical testing of expanded and activated NK cells for cell therapy
of cancer.
Natural killer (NK) cells can kill cancer cells in the absence of prior stimulation and hold considerable
potential for cell-based therapies targeting human malignancies (14). This notion is corroborated by
the observation that, among patients with leukemia undergoing hematopoietic stem cell
transplantation, the antileukemic effect of the transplant was significantly greater when the donor NK
cells exhibited a killer inhibitory receptor (KIR) profile that predicted a higher cytotoxicity against the
leukemic cells of the recipient (3;57). Moreover, allogeneic NK cells might be beneficial when directly
infused into patients, a procedure that was shown to induce clinical remission in patients with high-risk
acute myeloid leukemia (AML) (8). Infusions of NK cells have also been proposed as a means to
improve the treatment of other cancers (9).
Because NK cells represent a small fraction of peripheral blood mononuclear cells, generating them in
numbers sufficient to meet clinical requirements, especially if multiple infusions are planned, is
problematic. Hence, NK cell-based therapies would greatly benefit from reliable methods to produce
large numbers of fully functional NK cells ex vivo. Unlike T and B lymphocytes, which readily respond
to a variety of stimuli, NK cells typically do not undergo sustained proliferation. Indeed, their reported
proliferative responses to cytokines with or without coculture with other cells have generally been
modest and of short duration in most studies (1016).
We previously found that the K562 leukemia cell line genetically modified to express membrane-bound
interleukin (IL)-15 and 41BB ligand specifically activates NK cells, drives them into the cell cycle and
allows their genetic modification (17). In this study, we determined the capacity of NK cells stimulated
by contact with K562-mb15-41BBL cells to exert anti-AML cytotoxicity.

.
We found that K562-mb15-41BBL cells induce sustained and specific proliferation of human NK cells.
NK cell expansion was observed in all donors tested, including patients with acute leukemia
undergoing therapy, with no apparent proliferative advantage of any particular NK cell subset. Gene
expression of NKAES-NK cells was markedly different than that of unstimulated and IL-2-stimulated
cells, not only in regards to their expression of cell proliferation-associated genes but also in that of
molecules that might regulate NK-cell function and their interaction with other cell types. NKAES-NK
cells had powerful cytotoxicity against AML cell lines and AML cells from patients, and were more
potent than unstimulated or IL-2-activated NK cells from the same donors. Based on these findings,
and on the effectiveness of NKAES-NK cells in murine models of AML, we developed a Master Cell
Bank of K562-mb15-41BBL cells under cGMP guidelines, and demonstrated that large-scale expansion
and activation of human NK cells for clinical studies was feasible, producing expansions of
CD56+CD3 cells that were even higher than those observed in the initial small-scale experiments
while maintaining high anti-AML cytotoxicity.
IL-2 can induce proliferative responses in human NK cells but only a minor fraction sustains continued
growth (10;26;27). Conceivably, some NK-cell subsets might be more responsive, as suggested by
early reports of up to 50-fold expansion after culture with IL-2 for 2 weeks of an NK subset that
adheres to plastic (2831). It is unclear, however, whether some CD3 + cells might have had, at least in
part, contributed to the increased cell numbers (29;30). More recently, anti-CD3 and IL-2 reportedly
induced 190-fold NK expansions after 21 days from the blood of healthy individuals (32) and,
surprisingly, 1600-fold expansions after 20 days from that of patients with myeloma (25). However,
these cells cytotoxicity against K562 cells was <10% at 1 : 1 E : T (25), a ratio at which NKAES-NK
cells from healthy donors or leukemia patients had a median cytotoxicity of 69% cells. Our results with
IL-2 alone or in combination with other cytokines are in line with those of earlier reports
(10;26;27;33). Indeed, most investigators have indicated that sustained expansions of
CD56+CD3 cells require additional signals (14;16), such as the presence of B-lymphoblastoid cells
(26;34;35). B-lymphoblastoid cells, however, also induce vigorous expansions of T lymphocytes,
whereas NKAES cultures do not stimulate T-cell proliferation. In the setting of allogeneic NK-cell
therapy, this could be an important practical advantage as it would facilitate the complete removal of
residual T cells at the end of the cultures (to avoid the risk of graft-versus-host disease). Because
K562-mb15-41BBL cells are lethally-irradiated before culture and they are lysed by the expanding NK
cells, the risk of infusing viable K562-mb15-41BBL is negligible. Nevertheless, we have incorporated
safeguards in our clinical protocol. We prepare cultures of irradiated K562-mb15-41BBL cells, and

monitor their growth and DNA-synthesis rate. We also test for the presence of viable K562-mb1541BBL cells at the end of the culture by flow cytometry, using GFP as a marker. The clinical product is
released only if there is no cell growth and no viable of K562-mb15-41BBL cell at the end of the
cultures.
Most patients with AML respond to initial treatment and achieve remission, but occult resistant
leukemia persists in approximately half of the patients, leading to overt (and usually fatal) relapse
(36;37). NK cell infusions have shown to be clinically effective in patients with high-risk AML (8); they
are being considered for the therapy of other hematological malignancies (9;38). Conceivably, NK-cell
therapy will be most powerful when the number of NK cells infused is sufficiently high to produce a
high E : T ratio. In our murine models of AML, multiple injections of NKAES-derived cells were required
to eradicate leukemia and achieve long-term remissions. The number of NK cells that can be
generated with the method that we describe should meet the requirement for a high E : T ratio,
particularly in the setting of minimal residual disease, and allow multiple NK cell infusions. We found
that administration of IL-2 significantly prolonged the survival of NKAES-NK cells in immunodeficient
mice. It is possible that other cytokines not yet available for clinical studies, such as IL-15, might
prove to be superior for this purpose. Of note, it was shown in clinical studies that lymphodepletion of
the recipients, a procedure essential to ensure prolonged engraftment of the infused cells (39),
resulted in high levels of serum IL-15 (8).
Although infusion of allogeneic unstimulated or IL-2-stimulated NK cells has proven to be safe, with no
significant graft-versus-host disease detected, the safety of NKAES-NK cell infusions must be
established. To this end, we have begun a Phase I dose-escalation clinical study of haploidentical
NKAES-NK cells in patients with refractory leukemia. In addition to AML and other hematologic
malignancies, some solid tumors should also be susceptible to NK cell cytotoxicity (9). Therefore,
patients with these malignancies could also be eligible for clinical studies of NK cell therapy.
ADOPTIVE T CELL THERAPY: HARNESSING THE IMMUNE SYSTEM TO FIGHT CANCER
August 15, 2014 | by Hiu Chung So

http://www.cityofhope.org/blog/adoptive-t-cell-fight-cancer

Immunotherapy using ones immune system to treat a disease has been long lauded as the
magic bullet of cancer treatments, one that can be more effective than the conventional therapies of
surgery, radiation or chemotherapy. One specific type of immunotherapy, called adoptive T cell
therapy, is demonstrating promising results for blood cancers and may have potential against other
types of cancers, too.

In adoptive T cell therapy, T cells (in blue, above) are extracted from the patient and modified to
recognize unique cancer markers and attack the cells carrying those markers. They are
then reinfused back into the patient, where they can kill cancer cells throughout the body. (Photo
credit: Lawrence Berkeley Laboratory)
What is adoptive T cell therapy and how does it work to treat cancer?
Every day, our immune system works to recognize and destroy abnormal, mutated cells. But the
abnormal cells that eventually become cancer are the ones that slip past this defense system. The
idea behind this therapy is to make immune cells (specifically, T lymphocytes) sensitive to cancerspecific abnormalities so that malignant cells can be targeted and attacked throughout the body.
Who would be good candidates for this type of therapy?
Currently, adoptive T cell therapy is mostly used to treat lymphoma and lymphoidleukemia, because
these cancer cells have unique surface markers that we can reprogram T cells to recognize and attack.
However, we also studying how to adapt this approach to treat other cancers as well,
including myeloid leukemia, multiplemyeloma and solid tumors.
What happens to the patient during this therapy?
First, we collect the patients own T cells from the bloodstream, which takes about four hours. The
cells are then modified to recognize the patients cancer; a two- to three-week process in our
laboratories. They are then frozen for later use as needed.
While the T cells are being modified, the patient undergoes an autologous stem cell transplant.
Afterward, the re-engineered T cells are infused back into the patient so that they can kill any

residual cancer cells that remained after the transplant. Depending on the type of cancer, its stage,
the patients health and other factors, some patients may receive the modified T cell
infusions shortly after their transplant; others may get their infusions later on, when tests showed
that the cancer has relapsed.
.more

The Application of Natural Killer Cell Immunotherapy for the Treatment of Cancer
Katayoun Rezvani1* and Rayne H. Rouce2,3
THIS ARTICLE IS PART OF THE RESEARCH TOPIC

NK cell-based cancer immunotherapy

Front. Immunol., 17 November 2015 | http://dx.doi.org/10.3389/fimmu.2015.00578

Natural killer (NK) cells are essential components of the innate immune system and play a critical role
in host immunity against cancer. Recent progress in our understanding of NK cell immunobiology has
paved the way for novel NK cell-based therapeutic strategies for the treatment of cancer. In this
review, we will focus on recent advances in the field of NK cell immunotherapy, including
augmentation of antibody-dependent cellular cytotoxicity, manipulation of receptor-mediated
activation, and adoptive immunotherapy with ex vivo-expanded, chimeric antigen receptor (CAR)engineered, or engager-modified NK cells. In contrast to T lymphocytes, donor NK cells do not attack
non-hematopoietic tissues, suggesting that an NK-mediated antitumor effect can be achieved in the
absence of graft-vs.-host disease. Despite reports of clinical efficacy, a number of factors limit the
application of NK cell immunotherapy for the treatment of cancer, such as the failure of infused NK
cells to expand and persist in vivo. Therefore, efforts to enhance the therapeutic benefit of NK cellbased immunotherapy by developing strategies to manipulate the NK cell product, host factors, and
tumor targets are the subject of intense research. In the preclinical setting, genetic engineering
of NK cells to express CARs to redirect their antitumor specificity has shown significant
promise. Given the short lifespan and potent cytolytic function of mature NK cells, they are attractive
candidate effector cells toexpress CARs for adoptive immunotherapies. Another innovative
approach to redirect NK cytotoxicity towards tumor cells is to create either bispecific or trispecific
antibodies, thus augmenting cytotoxicity against tumor-associated antigens. These are
exciting times for the study of NK cells; with recent advances in the field of NK cell biology and

translational research, it is likely that NK cell immunotherapy will move to the forefront of cancer
immunotherapy over the next few years.
Natural killer (NK) cell-mediated cytotoxicity contributes to the innate immune response against
various malignancies, including leukemia (1, 2). The antitumor effect of NK cells is a subject of intense
investigation in the field of cancer immunotherapy. In this review, we will focus on recent advances
in NK cell immunotherapy, including

augmentation of antibody-dependent cytotoxicity,

manipulation of receptor-mediated activation, and

adoptive immunotherapy with ex vivo-expanded,

chimeric antigen receptor (CAR)-engineered, or

engager-modified NK cells.

Biology of NK Cells Relevant to Adoptive Immunotherapy

Natural killer cells are characterized by the lack of CD3/TCR molecules and by the expression of CD16
and CD56 surface antigens. Around 90% of circulating NK cells are CD56 dim, characterized by their
distinct ability to mediate cytotoxicity in response to target cell stimulation (3, 4). This subset includes
the alloreactive NK cells that play a central role in targeting leukemia cells in the setting of allogeneic
hematopoietic stem cell transplant (HSCT) (5). The remaining NK cells, predominantly housed in
lymphoid organs, are CD56bright, and although less mature (unlicensed) (3, 6, 7), they have a greater
capability to secrete and respond to cytokines (8, 9). CD56bright and CD56dim NK cells are also
distinguished by their differential expression of FcRIII (CD16), an integral determinant of NKmediated antibody-dependent cellular cytotoxicity (ADCC), with CD56 dim NK cells expressing high
levels of the receptor, while CD56bright NK cells are CD16 dim or negative (6). In contrast to T and B
lymphocytes, NK cells do not express rearranged, antigen-specific receptors; rather, NK effector
function is dictated by the integration of signals received through germ-line-encoded receptors that
can recognize ligands on their cellular targets. Functionally, NK cell receptors are classified as
activating or inhibitory. NK cell function, including cytotoxicity and cytokine release, is governed by a
balance between signals received from inhibitory receptors, notably the killer Ig-like receptors (KIRs)
and the heterodimeric C-type lectin receptor (NKG2A), and activating receptors, in particular the
natural cytotoxicity receptors (NCRs) NKp46, NKp30, NKp44, and the C-type lectin-like activating
immunoreceptor NKG2D (9).
The inhibitory KIRs (iKIRs) with known HLA ligands include KIR2DL2 and KIR2DL3, which recognize
the HLA-C group 1-related alleles characterized by an asparagine residue at position 80 of the -1
helix (HLA-CAsn80); KIR2DL1, which recognizes the HLA-C group 2-related alleles characterized by a

lysine residue at position 80 (HLA-CLys80); and KIR3DL1, which recognizes the HLA-Bw4 alleles
(9, 10). NK cells also express several activating receptors that are potentially specific for selfmolecules. KIR2DS1 has been shown to interact with group 2 HLA-C molecules (HLA-C2), while
KIR2DS2 was recently shown to recognize HLA-A*11 (10, 11). Hence, these receptors require
mechanisms to prevent inadvertent activation against normal tissues, processes referred to as
tolerance to self. Engagement of iKIR receptors by HLA class I leads to signals that block NK-cell
triggering during effector responses. These receptors explain the missing self hypothesis, which
postulates that NK cells survey tissues for normal levels of the ubiquitously expressed MHC class I
molecules (12, 13). Upon cellular transformation or viral infection, surface MHC class I expression on
the cell surface is often reduced or lost to evade recognition by antitumor T cells. When a mature NK
cell encounters transformed cells lacking MHC class I, their inhibitory receptors are not engaged, and
the unsuppressed activating signals, in turn, can trigger cytokine secretion and targeted attack of the
virus-infected or transformed cell (13, 14). In parallel, cellular stress and DNA damage (occurring in
cells during viral or malignant transformation) results in upregulation of stress ligands that can be
recognized by activating NK receptors. Thus, human tumor cells that have lost self-MHC class I
expression or bear altered-self stress-inducible proteins are ideal targets for NK recognition and
killing (1416). NK cells directly kill tumor cells through several mechanisms, including release of
cytoplasmic granules containing perforin and granzyme (1618), expression of tumor necrosis factor
(TNF) family members, such as FasL or TNF-related apoptosis-inducing ligand (TRAIL), which induce
tumor cell apoptosis by interacting with their respective receptors Fas and TRAIL receptor (TRAILR)
(1619) as well as ADCC (9).

Interaction Between Natural Killer Cells and Other Immune Subsets

Increasing understanding of NK cell biology and their interaction with other cells of the immune
system has led to several novel immunotherapeutic approaches as discussed in this review. NK cells
produce cytokines that can exert regulatory control of downstream adaptive immune responses by
influencing the magnitude of T cell responses, specifically T helper-1 (TH1) function (20). NK cell
function, in turn, is regulated by cytokines, such as IL-2, IL-15, IL-12, and IL-18 (21), as well as by
interactions with other cell types, such as dendritic cells, macrophages, and mesenchymal stromal
cells (10, 22, 23). IL-15 has emerged as a pivotal cytokine required for NK cell development and
maintenance. Whereas mice deficient in IL-2 (historically the cytokine of choice to expand and activate
NK cells) have normal NK cells, IL-15-deficient mice lack NK cells (24).

Several cytokines are also known to inhibit NK cell activation and function, thus playing a crucial role
in tumor escape from NK immune surveillance. Recently, considerable attention has been paid to the
inhibitory effects of transforming growth factor-beta (TGF-) and IL-10 on NK cell cytotoxicity
(12, 25, 26). Several groups have shown that secretion of TGF- by tumor cells results in
downregulation of activating receptors, such as NKp30 and NKG2D, with resultant NK dysfunction
(25,26). Similarly, IL-10 production by acute myeloid leukemia (AML) blasts induces upregulation of
NKG2A with significant impairment in NK function (3).

Modulation of Antibody-Dependent Cellular Cytotoxicity

The CD56dim subset of NK cells expresses the Fc receptor CD16, through which NK cells mount ADCC,
providing opportunities for its modulation to augment NK effector function (27, 28). In fact, a number
of clinically approved therapeutic antibodies targeting tumor-associated antigens (such as rituximab or
cetuximab) function at least partially through triggering NK cell-mediated ADCC. Several studies using
mouse tumor models have established that efficient antibodyFc receptor (FcR) interactions are
essential for the efficacy of monoclonal antibody (mAb) therapy, a mainstay of cancer therapy
(28, 29). Based on this premise, Romain et al. successfully engineered the Fc region of the IgG mAb,
HuM195 targeting the AML leukemia antigen CD33, by introducing the triple mutation
S293D/A330L/I332E (DLE). Using timelapse imaging microscopy in nanowell grids (TIMING, a method
of analyzing kinetics of thousands of NK cells and mAb-coated targets), they demonstrated that the
DLE-HuM195 antibody increased both the quality and quantity of NK cell-mediated ADCC by recruiting
NK cells to participate in cytotoxicity via CD16-mediated signaling. NK cells encountering DLEHuM195-coated targets induced rapid target cell apoptosis by promoting conjugation to multiple target
cells (leading to increased serial killing of targets), thus inducing apoptosis in twice the number of
targets as the wild-type mAb (27).
Additional approaches under investigation to enhance NK cell-mediated ADCC include antibody
engineering and therapeutic combination of antibodies predicted to have synergistic activity. For
example, mogamulizumab (an anti-CCR4 mAb recently approved in Japan) is defucosylated to
increase binding by FcRIIIA and thereby enhances ADCC. Mogamulizumab successfully induced ADCC
activity against CCR4-positive cell lines and inhibited the growth of EBV-positive NK-cell lymphomas in
a murine xenograft model (30). These findings suggest that mogamulizumab may be a therapeutic
option against EBV-associated T and NK-lymphoproliferative diseases (30). Obinutuzumab (GA101) is
a novel type II glycoengineered mAb against CD20 with increased FcRIII binding and ADCC activity.
In contrast to rituximab, GA101 induces activation of NK cells irrespective of their inhibitory KIR

expression, and its activity is not negatively affected by KIR/HLA interactions (31). These data show
that modification of the Fc fragment to enhance NK-mediated ADCC can be an effective strategy to
augment the efficacy of therapeutic mAbs (31).
Although enhanced NK-mediated ADCC occurs in the presence of certain mAbs, in the case of nonengineered mAbs (such as rituximab), this NK-mediated cytotoxicity is typically still under the
jurisdiction of KIR-mediated inhibition. However, ADCC responses can be potentiated in vitro in the
presence of antibodies that block NK cell inhibitory receptor interaction with MHC class I ligands (32).
These include the use of anti-KIR Abs to block the interaction of iKIRs with their cognate HLA class I
ligands. To exploit this pathway pharmacologically, a fully humanized anti-KIR mAb 1-7F9 (IPH2101)
(33) with the ability to block KIR2DL1/L2/L3 and KIR2DS1/S2 was generated. In vitro, anti-KIR mAbs
can augment NK cell-mediated lysis of HLA-C-expressing tumor cells, including autologous AML blasts
and autologous CD138+ multiple myeloma (MM) cells (34). Additionally, in a dose-escalation phase 1
clinical trial in elderly patients with AML, 1-7F9 mAb was reported to be safe and could block KIRs for
prolonged periods (35). A recombinant version of this mAb with a stabilized hinge (lirilumab) was
recently developed. Lirilumab is a fully humanized IgG4 anti-KIR2DL1, -L2, -L3, -S1, and -S2 mAb.
The iKIRs targeted by lirilumab collectively recognize virtually all HLA-C alleles, and the blockade of
the three KIR2DLs allows targeting of every patient without the need for prior HLA or KIR typing
(33, 34). Furthermore, the combination of an anti-KIR mAb with the immunomodulatory drug
lenalidomide was shown to potentiate ADCC and is being tested in a phase 1 clinical trial in patients
with MM [NCT01217203 (35)]. A potential concern is related to how inhibitory KIR blockade may
impact on the ability of NK cells to discriminate self, healthy cells from abnormal virally infected or
cancerous cells. Preliminary in vitrodata suggest that Ab blockade of iKIRs will preferentially augment
the ADCC response, without increasing cytotoxicity against self healthy cells (32). It is reassuring that
in the IPH2101 phase 1 studies, no alterations in the expression of major inhibitory or activating NK
receptors or frequencies of circulating peripheral lymphocytes were reported, indicating that the Ab
does not induce clinically significant targeting of normal cells by NK cells (35). Lin et al. recently
reported on the application of an agonistic NK cell-targeted mAb to augment ADCC (36). Following FcR
triggering during ADCC, expression of the activation marker CD137 is increased. Agonistic antibodies
targeting CD137 have been reported to augment NK-cell function, including degranulation, secretion of
IFN-, and antitumor cytotoxicity in in vitro and in vivo preclinical models of tumor (3639). The
combination of the agonistic anti-CD137 antibody with rituximab is currently being evaluated in a
phase 1 trial in patients with lymphoma [NCT01307267 (3537)].

Other factors, such as specific CD16 polymorphisms and NKG2D engagement, can also influence
ADCC, with certain polymorphisms (such as FcRIIIa-V158F polymorphism) resulting in a stronger IgG
binding (40). These findings are clinically relevant, as supported by the observation that patients with
non-Hodgkin lymphoma (NHL) with the FcRIIIa-V158F polymorphism experienced improved clinical
response to rituximab (41, 42). In summary, several antibody combinations designed to boost ADCC
have shown promising results in preclinical and early clinical trials, thus warranting further study of
this strategy to enhance NK cell activity against tumor cells.

Adoptive Transfer of Autologous NK Cells

The early studies of adoptive NK cell therapy focused on enhancing the antitumor activity of
endogenous NK cells (43). Initial trials of adoptive NK therapy in the autologous setting involved using
CD56 beads to select NK cells from a leukapheresis product and subsequently infusing the beadselected autologous NK cells into patients (43, 44). Infusions were followed by administration of
systemic cytokines (most commonly IL-2) to provide additional in vivo stimulation and support their
expansion. This strategy met with limited success due to a combination of factors (44). Although
cytokine stimulation promoted NK cell activation and resulted in greater cytotoxicity against malignant
targets in vitro, only limited in vivoantitumor activity was observed (4345). Similar findings were
observed when autologous NK cells and systemic IL-2 were given as consolidation treatment to
patients with lymphoma who underwent autologous BMT (46). The poor clinical outcomes observed
with adoptive transfer of ex vivo activated autologous NK cells followed by systemic IL-2 were
attributed to three factors: (1) development of severe life-threatening side effects, such as vascular
leak syndrome as a result of IL-2 therapy; (2) IL-2-induced expansion of regulatory T cells known to
directly inhibit NK cell function and induce activation-induced cell death (4749); and (3) lack of
antitumor effect related to the inhibition of autologous NK cells by self-HLA molecules. Strategies to
overcome this autologous checkpoint, thus redirecting autologous NK cells to target and kill leukemic
blasts are the subject of intense investigation (3335). These include the use of anti-KIR Abs (such as
the aforementioned lirilumab) to block the interaction of inhibitory receptors on the surface of NK cells
with their cognate HLA class I ligand.

Exploiting the Alloreactivity of Allogeneic NK Cells Adoptive Immunotherapy and

Beyond

An alternative strategy is to use allogeneic instead of autologous NK cells, thus taking advantage of
the inherent alloreactivity afforded by the missing self concept (13). Over the past decade, adoptive
transfer of ex vivo-activated or -expanded allogeneic NK cells has emerged as a promising
immunotherapeutic strategy for cancer (24, 5052). Allogeneic NK cells are less likely to be subject to
the inhibitory response resulting from NK cell recognition of self-MHC molecules as seen with
autologous NK cells. A number of studies have shown that infusion of haploidentical NK cells to exploit
KIR/HLA alloreactivity is safe and can mediate impressive clinical activity in some patients with AML
(5052). In fact, algorithms have been developed to ensure selection of stem cell donors with the
greatest potential for NK cell alloreactivity for allogeneic HSCT (50).
Promising results in the HSCT setting suggest that the application of this strategy in the nontransplant setting may be a plausible option. Miller et al. were among the first to show that adoptive
transfer of ex vivo-expanded haploidentical NK cells after lymphodepleting chemotherapy is safe, and
can result in expansion of NK cells in vivo without inducing graft-vs.-host disease (GVHD) (50). In a
phase I dose-escalation trial, 43 patients with either hematologic malignancies (poor prognosis AML or
Hodgkin lymphoma) or solid tumor (metastatic melanoma or renal cell carcinoma) received up to 2
107cells/kg of haploidentical NK cells following either low intensity [low-dose cyclophosphamide (Cy)
and methylprednisolone or fludarabine (Flu)] or high intensity regimens (Hi-Cy/Flu). All patients
received subcutaneous IL-2 after NK cell infusion. Whereas adoptively infused NK cells persisted only
transiently following low intensity regimens, AML patients who received the more intense Hi-Cy/Flu
regimen had a marked rise in endogenous IL-15 associated with expansion of donor NK cells and
induction of complete remission (CR) in five of 19 very high-risk patients. The superior NK expansion
observed after high-dose compared to low-dose chemotherapy was attributed to a combination of
factors including prevention of host T cell-mediated rejection and higher levels of cytokines, such as
IL-15. These findings provided the first evidence that haploidentical NK cells are safe and can persist
and expand in vivo, supporting the proof of concept that NK cells may be applied for the treatment of
selected malignancies either alone or as an adjunct to HSCT (50).
Another pivotal pilot study, the NKAML trial (Pilot Study of Haploidentical NK Transplantation for AML),
reported that infusion of KIR-HLA-mismatched donor NK cells can reduce the risk of relapse in
childhood AML (51). Ten pediatric patients with favorable or intermediate risk AML in first CR were
enrolled following completion of 45 cycles of chemotherapy. All patients received a low-dose
conditioning regimen consisting of Cy/Flu prior to infusion of NK cells (median, 29 10 6/kg NK cells)
from a haploidentical donor, followed by six doses of IL-2. NK infusions were well tolerated with limited
non-hematologic toxicity. All patients had transient engraftment of NK cells for a median of 10 days

(range 2189 days) with significant expansion of KIR-mismatched NK cells. With a median follow-up of
964 days, all patients remained in remission, suggesting that donor-recipient HLA-mismatched NK
cells may reduce the risk of relapse in childhood AML (51).
Other strategies currently under investigation include the infusion of KIR-ligand-mismatched
haploidentical NK cells as part of the pre-HSCT conditioning regimen (NCT00402558), and NK cell
infusion to prevent relapse or as therapy for minimal residual disease in patients after haploidentical
HSCT (NCT01386619).
.more

Human NK Cell Lines as a Source of NK Immunotherapy

The adoptive transfer of NK cell lines has several theoretical advantages over the use of patient- or
donor-derived NK cells. These are primarily related to the lack of expression of iKIRs, presumed lack
of immunogenicity, ease of expansion and availability as an off-the-shelf product (85). Several
human NK cell lines, such as NK-92 and KHYG-1, have been documented to exert antitumor activity in
both preclinical and clinical settings (8688). NK-92, the most extensively characterized NK-cell line,
was established in 1994 from the PB of a male Caucasian patient with NHL. NK-92 cells are IL-2dependent, harbor a CD2+CD56+CD57+ phenotype and exert potent in vitro cytotoxicity (86). Infusion
of up to 1010 cells/m2NK-92 cells into patients with advanced lung cancer and other advanced
malignancies was well tolerated and the cells persisted for a minimum of 48 h with encouraging clinical
responses (86, 8891). However, potential limitations of using NK cell lines, such as NK-92 cells,
include the requirement for irradiation to reduce the risk of engrafting cells with potential in
vivo tumorigenicity, and the need for pre-infusion conditioning to avoid host rejection. Furthermore,
infusion of allogeneic NK cell lines may induce T and B cell alloimmune responses, limiting their in
vivo persistence and precluding multiple infusions. A number of studies are testing NK-92 cells
(Neukoplast) in patients with solid tumors, such as Merkel cell cancer and renal cell carcinoma, as
well as in hematological malignancies (85).
While results from clinical studies of NK cell adoptive therapy are encouraging (4852, 70), significant
gaps remain in our understanding of the optimal conditions for NK cell infusion. Based on the
pioneering work from Rosenberg et al. demonstrating the importance of lymphodepletion to support
the expansion of tumor-infiltrating T cells (92) and given its emergence as a key determinant of
efficacy with CAR therapy, several groups are actively investigating the ideal preparative regimen to
promote the expansion and persistence of adoptively infused NK cells (53, 69, 70, 75). Available data

support the use of high-dose Cy/Flu regimen as the frontrunner, considering it is reasonably well
tolerated and shown to support the in vivo expansion of NK cells (51, 70). IL-15 is an ideal candidate
cytokine for the expansion of NK cells in vivo, especially since it does not promote expansion of
regulatory T cells (66), which have been shown to suppress NK cell effector function in IL-2-based
trials (69, 70). In a recent phase 1 study in patients with metastatic melanoma or renal cell
carcinoma, rhIL-15 was shown to activate NK cells, monocytes, , and CD8 T cells (93). However, as
an intravenous bolus dose, rhIL-15 proved too difficult to administer because of significant clinical
toxicities (93). Based on these promising data, alternative dosing strategies are being investigated,
including continuous intravenous infusions. To this effect, systemic IL-15 along with infusion of donor
NK cells are currently being tested in a phase I clinical trial for AML (NCT01385423).
..

Bispecific and Trispecific Engagers

An innovative immunoglobulin-based strategy to redirect NK cytotoxicity towards tumor cells is to
create either bispecific or trispecific antibodies (BiKE, TriKE) (113). BiKEs are constructed by joining a
single-chain Fv against CD16 and a single-chain Fv against a tumor-associated antigen (BiKE), or two
tumor-associated antigens (TriKE). Gleason et al. showed that bispecific (bscFv) CD16/CD19 and
trispecific (tscFv) CD16/CD19/CD22 engagers directly trigger NK cell activation through CD16,
significantly increasing NK cell cytolytic activity and cytokine production against various CD19expressing B cell lines. The same group also developed and tested a CD16 33 BiKE in refractory AML
and demonstrated that the potent killing by NK cells could overcome the inhibitory effect of KIR
signaling (113, 114).
Notably, activated NK cells lose CD16 (FcRIII) and CD62L through a metalloprotease called ADAM17,
which is expressed on NK cells, which may in turn impact on the efficacy of Fc-mediated cytotoxicity
(115). Romee et al. recently showed that selective inhibition of ADAM17 enhances CD16-mediated NK
cell function by preserving CD16 on the NK cell surface, thus enhancing ADCC (115). Additionally, Fcinduced production of cytokines by NK cells exposed to rituximab-coated B cell targets can be further
enhanced by ADAM17 inhibition. These findings support a role for targeting ADAM17 to prevent CD16
shedding and to improve the efficacy of therapeutic mAbs. The same group subsequently discovered
that ADAM17 inhibition enhances CD16 33 BiKE responses against primary AML targets (114).

NK Cells What Does the Future Hold?

Recent advances in the understanding of NK cell immunobiology have paved the way for novel and
innovative anti-cancer therapies. Here, we have discussed a representation of these novel
immunotherapeutic strategies to potentiate NK cell function and enhance antitumor activity including
ADCC-inducing mAbs, ex vivoactivated or genetically modified NK cells and bi- or trispecific engagers
(Figure 1).
..

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Microbe meets cancer

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microbiome, gut microbiota,immunotherapy, Microbiology, microbiome on April 10, 2016 | Leave a Comment

2 Votes

Microbe meets cancer

Larry H. Bernstein, MD, FCAP, Curator
LPBI

Microbes Meet Cancer

Understanding cancers relationship with the human microbiome could transform immune-modulating
therapies.

By Kate Yandell | April 1, 2016 http://www.the-scientist.com/?

articles.view/articleNo/45616/title/Microbes-Meet-Cancer

ISTOCK.COM/KATEJA_FN; ISTOCK.COM/FRANK RAMSPOTT http://www.thescientist.com/images/April2016/feature1.jpg

In 2013, two independent teams of scientists, one in Maryland and one in France, made a surprising
observation: both germ-free mice and mice treated with a heavy dose of antibiotics responded poorly
to a variety of cancer therapies typically effective in rodents. The Maryland team, led by Romina
Goldszmidand Giorgio Trinchieri of the National Cancer Institute, showed that both an investigational
immunotherapy and an approved platinum chemotherapy shrank a variety of implanted tumor types
and improved survival to a far greater extent in mice with intact microbiomes. 1 The French group, led
by INSERMs Laurence Zitvogel, got similar results when testing the long-standing chemotherapeutic
agent cyclophosphamide in cancer-implanted mice, as well as in mice genetically engineered to
develop tumors of the lung.2
The findings incited a flurry of research and speculation about how gut microbes contribute to cancer
cell death, even in tumors far from the gastrointestinal tract. The most logical link between the
microbiome and cancer is the immune system. Resident microbes can either dial up inflammation or
tamp it down, and can modulate immune cells vigilance for invaders. Not only does the immune
system appear to be at the root of how the microbiome interacts with cancer therapies, it also appears
to mediate how our bacteria, fungi, and viruses influence cancer development in the first place.
We clearly see shifts in the [microbial] community that precede development of tumors, says
microbiologist and immunologist Patrick Schloss, who studies the influence of the microbiome on colon
cancer at the University of Michigan.

But the relationship between the microbiome and cancer is complex: while some microbes promote
cell proliferation, others appear to protect us against cancerous growth. And in some cases, the
conditions that spur one cancer may have the opposite effect in another. Its become pretty obvious
that the commensal microbiota affect inflammation and, through that or through other mechanisms,
affect carcinogenesis, says Trinchieri. What we really need is to have a much better understanding of
which species, which type of bug, is doing what and try to change the balance.
Gut feeling
In the late 1970s, pathologist J. Robin Warren of Royal Perth Hospital in Western Australia began to
notice that curved bacteria often appeared in stomach tissue biopsies taken from patients with chronic
gastritis, an inflammation of the stomach lining that often precedes the development of stomach
cancer. He and Barry J. Marshall, a trainee in internal medicine at the hospital, speculated that the
bacterium, now called Helicobacter pylori, was somehow causing the gastritis. 3 So committed was
Marshall to demonstrating the microbes causal relationship to the inflammatory condition that he had
his own stomach biopsied to show that it contained no H. pylori, then infected himself with the
bacterium and documented his subsequent experience of gastritis. 4 Scientists now accept that H.
pylori, a common gut microbe that is present in about 50 percent of the worlds population, is
responsible for many cases of gastritis and most stomach ulcers, and is a strong risk factor for
stomach cancer.5 Marshall and Warren earned the 2005 Nobel Prize in Physiology or Medicine for their
work.
H. pylori may be the most clear-cut example of a gut bacterium that influences cancer development,
but it is likely not the only one. Researchers who study cancer in mice have long had anecdotal
evidence that shifts in the microbiome influence the development of diverse tumor types. You have a
mouse model of carcinogenesis. It works beautifully, says Trinchieri. You move to another institution.
It works completely differently, likely because the animals microbiomes vary with environment.

IMMUNE INFLUENCE: In recent years, research has demonstrated that microbes living in and on the
mammalian body can affect cancer risk, as well as responses to cancer treatment. Although the details
of this microbe-cancer link remain unclear, investigators suspect that the microbiomes ability to
modulate inflammation and train immune cells to react to tumors is to blame.
See full infographic: WEB | PDF AL GRANBERG
Around the turn of the 21st century, cancer researchers began to systematically experiment with the
rodent microbiome, and soon had several lines of evidence linking certain gut microbes with a mouses
risk of colon cancer. In 2001, for example, Shoichi Kado of the Yakult Central Institute for
Microbiological Research in Japan and colleagues found that a strain of immunocompromised mice
rapidly developed colon tumors, but that germ-free versions of these mice did not. 6 That same year,
an MIT-based group led by the late David Schauer demonstrated that infecting mice with the
bacterium Citrobacter rodentium spurred colon tumor development.7 And in 2003, MITs Susan
Erdman and her colleagues found that they could induce colon cancer in immunocompromised mice by
infecting them with Helicobacter hepaticus, a relative of? H. pylori that commonly exists within the
murine gut microbiome.8
More recent work has documented a similar link between colon cancer and the gut microbiome in
humans. In 2014, a team led by Schloss sequenced 16S rRNA genes isolated from the stool of 90
people, some with colon cancer, some with precancerous adenomas, and still others with no
disease.9 The researchers found that the feces of people with cancer tended to have an altered
composition of bacteria, with an excess of the common mouth
microbes Fusobacterium orPorphyromonas. A few months later, Peer Bork of the European Molecular

Biology Laboratory performed metagenomic sequencing of stool samples from 156 people with or
without colorectal cancer. Bork and his colleagues found they could predict the presence or absence of
cancer using the relative abundance of 22 bacterial species,
including Porphyromonas andFusobacterium.10 They could also use the method to predict colorectal
cancer with about the same accuracy as a blood test, correctly identifying about 50 percent of cancers
while yielding false positives less than 10 percent of the time. When the two tests were combined,
they caught more than 70 percent of cancers.
Whether changes in the microbiota in colon cancer patients are harbingers of the disease or a
consequence of tumor development remained unclear. What comes first, the change in the
microbiome or tumor development? asks Schloss. To investigate this question, he and his colleagues
treated mice with microbiome-altering antibiotics before administering a carcinogen and an
inflammatory agent, then compared the outcomes in those animals and in mice that had received only
the carcinogenic and inflammatory treatments, no antibiotics. The antibiotic-treated animals had
significantly fewer and smaller colon tumors than the animals with an undisturbed microbiome,
suggesting that resident bacteria were in some way promoting cancer development. And when the
researchers transferred microbiota from healthy mice to antibiotic-treated or germ-free mice, the
animals developed more tumors following carcinogen exposure. Sterile mice that received microbiota
from mice already bearing malignancies developed the most tumors of all. 11
Most recently, Schloss and his colleagues showed that treating mice with seven unique combinations
of antibiotics prior to exposing them to carcinogens yielded variable but predictable levels of tumor
formation. The researchers determined that the number of tumors corresponded to the unique ways
that each antibiotic cocktail modulated the microbiome. 12
Weve kind of proven to ourselves, at least, that the microbiome is involved in colon cancer, says
Schloss, who hypothesizes that gut bacteriadriven inflammation is to blame for creating an
environment that is hospitable to tumor development and growth. Gain or loss of certain components
of the resident bacterial community could lead to the release of reactive oxygen species, damaging
cells and their genetic material. Inflammation also involves increased release of growth factors and
blood vessel proliferation, potentially supporting the growth of tumors. (See illustration above.)
Recent research has also yielded evidence that the gut microbiota impact the development of cancer
in sites far removed from the intestinal tract, likely through similar immune-modulating mechanisms.
Systemic effects

In the mid-2000s, MITs Erdman began infecting a strain of mice predisposed to intestinal tumors
withH. hepaticus and observing the subsequent development of colon cancer in some of the animals.
To her surprise, one of the mice developed a mammary tumor. Then, more of the mice went on to
develop mammary tumors. This told us that something really interesting was going on, Erdman
recalls. Sure enough, she and her colleagues found that mice infected with H. hepaticus were more
likely to develop mammary tumors than mice not exposed to the bacterium. 13The researchers showed
that systemic immune activation and inflammation could contribute to mammary tumors in other, less
cancer-prone mouse models, as well as to the development of prostate cancer.

MICROBIAL STOWAWAYS: Bacteria of the human gut microbiome are intimately involved in cancer
development and progression, thanks to their interactions with the immune system. Some microbes,
such as Helicobacter pylori, increase the risk of cancer in their immediate vicinity (stomach), while
others, such as someBacteroides species, help protect against tumors by boosting T-cell
infiltration.EYE OF SCIENCE/SCIENCE SOURCE
http://www.the-scientist.com/images/April2016/immune_2.jpg

 DR. GARY GAUGLER/SCIENCE SOURCE http://www.thescientist.com/images/April2016/immune3.jpg

At the University of Chicago, Thomas Gajewski and his colleagues have taken a slightly different
approach to studying the role of the microbiome in cancer development. By comparing Black 6 mice
coming from different vendorsTaconic Biosciences (formerly Taconic Farms) and the Jackson
LaboratoryGajewski takes advantage of the fact that the animals different origins result in different
gut microbiomes. We deliberately stayed away from antibiotics, because we had a desire to model
how intersubject heterogeneity [in cancer development] might be impacted by the commensals they
happen to be colonized with, says Gajewski in an email to The Scientist.
Last year, the researchers published the results of a study comparing the progression of melanoma
tumors implanted under the mices skin, finding that tumors in the Taconic mice grew more
aggressively than those in the Jackson mice. When the researchers housed the different types of mice
together before their tumors were implanted, however, these differences disappeared. And
transferring fecal material from the Jackson mice into the Taconic mice altered the latters tumor
progression.14
Instead of promoting cancer, in these experiments the gut microbiome appeared to slow tumor
growth. Specifically, the reduced tumor growth in the Jackson mice correlated with the presence of
Bifidobacterium, which led to the greater buildup of T?cells in the Jackson mices
tumors. Bifidobacteriaactivate dendritic cells, which present antigens from bacteria or cancer cells to
T?cells, training them to hunt down and kill these invaders. Feeding Taconic mice bifidobacteria
improved their response to the implanted melanoma cells.

One hypothesis going into the experiments was that we might identify immune-suppressive bacteria,
or commensals that shift the immune response towards a character that was unfavorable for tumor
control, says Gajewski. But in fact, we found that even a single type of bacteria could boost the
antitumor immune response.

http://www.the-scientist.com/images/April2016/immune4.jpg

Drug interactions
Ideally, the immune system should recognize cancer as invasive and nip tumor growth in the bud. But
cancer cells display self molecules that can inhibit immune attack. A new type of immunotherapy,
dubbed checkpoint inhibition or blockade, spurs the immune system to attack cancer by blocking
either the tumor cells surface molecules or the receptors on T?cells that bind to them.

CANCER THERAPY AND THE MICROBIOME

In addition to influencing the development and progression of cancer by regulating inflammation
and other immune pathways, resident gut bacteria appear to influence the effectiveness of many
cancer therapies that are intended to work in concert with host immunity to eliminate tumors.

Some cancer drugs, such as oxaliplatin chemotherapy and CpG-oligonucleotide

immunotherapy, work by boosting inflammation. If the microbiome is altered in such a
way that inflammation is reduced, these therapeutic agents are less effective.
Cancer-cell surface proteins bind to receptors on T cells to prevent them from killing

cancer cells. Checkpoint inhibitors that block this binding of activated T cells to cancer
cells are influenced by members of the microbiota that mediate these same cell
interactions.
Cyclophosphamide chemotherapy disrupts the gut epithelial barrier, causing the gut to
leak certain bacteria. Bacteria gather in lymphoid tissue just outside the gut and spur
generation of T helper 1 and T helper 17 cells that migrate to the tumor and kill it.

As part of their comparison of Jackson and Taconic mice, Gajewski and his colleagues decided to test a
type of investigational checkpoint inhibitor that targets PD-L1, a ligand found in high quantities on the
surface of multiple types of cancer cells. Monoclonal antibodies that bind to PD-L1 block the PD-1
receptors on T?cells from doing so, allowing an immune response to proceed against the tumor cells.
While treating Taconic mice with PD-L1targeting antibodies did improve their tumor responses, they
did even better when that treatment was combined with fecal transfers from Jackson mice, indicating
that the microbiome and the immunotherapy can work together to take down cancer. And when the
researchers combined the anti-PD-L1 therapy with a bifidobacteria-enriched diet, the mices tumors
virtually disappeared.14
Gajewskis group is now surveying the gut microbiota in humans undergoing therapy with checkpoint
inhibitors to better understand which bacterial species are linked to positive outcomes. The
researchers are also devising a clinical trial in which they will give Bifidobacterium supplements to
cancer patients being treated with the approved anti-PD-1 therapy pembrolizumab (Keytruda), which
targets the immune receptor PD-1 on T?cells, instead of the cancer-cell ligand PD-L1.
Meanwhile, Zitvogels group at INSERM is investigating interactions between the microbiome and
another class of checkpoint inhibitors called CTLA-4 inhibitors, which includes the breakthrough
melanoma treatment ipilimumab (Yervoy). The researchers found that tumors in antibiotic-treated and
germ-free mice had poorer responses to a CTLA-4targeting antibody compared with mice harboring
unaltered microbiomes.15 Particular Bacteroides species were associated with T-cell infiltration of
tumors, and feedingBacteroides fragilis to antibiotic-treated or germ-free mice improved the animals
responses to the immunotherapy. As an added bonus, treatment with these
immunogenic Bacteroides species decreased signs of colitis, an intestinal inflammatory condition that
is a dangerous side effect in patients using checkpoint inhibitors. Moreover, Zitvogel and her
colleagues showed that human metastatic melanoma patients treated with ipilimumab tended to have
elevated levels of B. fragilis in their microbiomes. Mice transplanted with feces from patients who
showed particularly strong B. fragilis gains did better on anti-CTLA-4 treatment than did mice
transplanted with feces from patients with normal levels ofB. fragilis.

There are bugs that allow the therapy to work, and at the same time, they protect against colitis,
says Trinchieri. That is very exciting, because not only [can] we do something to improve the therapy,
but we can also, at the same time, try to reduce the side effect.
And these checkpoint inhibitors arent the only cancer therapies whose effects are modulated by the
microbiome. Trinchieri has also found that an immunotherapy that combines antibodies against
interleukin-10 receptors with CpG oligonucleotides is more effective in mice with unaltered
microbiomes.1He and his NCI colleague Goldszmid further found that the platinum chemotherapy
oxaliplatin (Eloxatin) was more effective in mice with intact microbiomes, and Zitvogels group has
shown that the chemotherapeutic agent cyclophosphamide is dependent on the microbiota for its
proper function.
Although the mechanisms by which the microbiome influences the effectiveness of such therapies
remains incompletely understood, researchers once again speculate that the immune system is the
key link. Cyclophosphamide, for example, spurs the body to generate two types of T?helper cells, T?
helper 1 cells and a subtype of T?helper 17 cells referred to as pathogenic, both of which destroy
tumor cells. Zitvogel and her colleagues found that, in mice with unaltered microbiomes, treatment
with cyclophosphamide works by disrupting the intestinal mucosa, allowing bacteria to escape into the
lymphoid tissues just outside the gut. There, the bacteria spur the body to generate T?helper 1 and T?
helper 17 cells, which translocate to the tumor. When the researchers transferred the pathogenic T?
helper 17 cells into antibiotic-treated mice, the mices response to chemotherapy was partly restored.

Microbiome modification
As the link between the microbiome and cancer becomes clearer, researchers are thinking about how
they can manipulate a patients resident microbial communities to improve their prognosis and
treatment outcomes. Once you figure out exactly what is happening at the molecular level, if there is
something promising there, I would be shocked if people dont then go in and try to modulate the
microbiome, either by using pharmaceuticals or using probiotics, says Michael Burns, a postdoc in the
lab of University of Minnesota genomicist Ran Blekhman.
Even if researchers succeed in identifying specific, beneficial alterations to the microbiome, however,
molding the microbiome is not simple. Its a messy, complicated system that we dont understand,
says Schloss.
So far, studies of the gut microbiome and colon cancer have turned up few consistent differences
between cancer patients and healthy controls. And the few bacterial groups that have repeatedly

shown up are not present in every cancer patient. We should move away from saying, This is a
causal species of bacteria, says Blekhman. Its more the function of a community instead of just a
single bacterium.
But the study of the microbiome in cancer is young. If simply adding one type of microbe into a
persons gut is not enough, researchers may learn how to dose people with patient-specific
combinations of microbes or antibiotics. In February 2016, a team based in Finland and China showed
that a probiotic mixture dubbed Prohep could reduce liver tumor size by 40 percent in mice, likely by
promoting an anti-inflammatory environment in the gut. 16
If it is true that, in humans, we can alter the course of the disease by modulating the composition of
the microbiota, says Jos Conejo-Garcia of the Wistar Institute in Philadelphia, thats going to be
very impactful.
Kate Yandell has been a freelance writer living Philadelphia, Pennsylvania. In February she
became an associate editor at Cancer Today.

GENETIC CONNECTION
The microbiome doesnt act in isolation; a patients genetic background can also greatly
influence response to therapy. Last year, for example, the Wistar InstitutesJos GarciaConejo and Melanie Rutkowski, now an assistant professor at the University of Virginia, showed
that a dominant polymorphism of the gene for the innate immune protein toll-like receptor 5
(TLR5) influences clinical outcomes in cancer patients by changing how the patients immune
cells interact with their gut microbes (Cancer Cell, 27:27-40, 2015).
More than 7 percent of people carry a specific mutation in TLR5 that prevents them from
mounting a full immune response when exposed to bacterial flagellin. Analyzing both genetic
and survival data from the Cancer Genome Atlas, Conejo-Garcia, Rutkowski, and their
colleagues found that estrogen receptorpositive breast cancer patients who carry
the TLR5 mutation, called the R392X polymorphism, have worse outcomes than patients without
the mutation. Among patients with ovarian cancer, on the other hand, those with
the TLR5 mutation were more likely to live at least six years after diagnosis than patients who
dont carry the mutation.
Investigating the mutations contradictory effects, the researchers found that mice with
normal TLR5produce higher levels of the cytokine interleukin 6 (IL-6) than those carrying the
mutant version, which have higher levels of a different cytokine called interleukin 17 (IL-17).
But when the researchers knocked out the animals microbiomes, these differences in cytokine
production disappeared, as did the differences in cancer progression between mutant and wild-

type animals.
The effectiveness of depleting specific populations or modulating the composition of the
microbiome is going to affect very differently people who are TLR5-positive or TLR5-negative,
says Conejo-Garcia. And Rutkowski speculates that many more polymorphisms linked to cancer
prognosis may act via microbiomeimmune system interactions. I think that our paper is just
the tip of the iceberg.

References
1.

N. Iida et al., Commensal bacteria control cancer response to therapy by

modulating the tumor microenvironment, Science, 342:967-70, 2013.

2.

S. Viaud et al., The intestinal microbiota modulates the anticancer immune effects
of cyclophosphamide, Science, 342:971-76, 2013.

3.

J.R. Warren, B. Marshall, Unidentified curved bacilli on gastric epithelium in active

chronic gastritis,Lancet, 321:1273-75, 1983.

4.

B.J. Marshall et al., Attempt to fulfil Kochs postulates for pyloric

Campylobacter, Med J Aust, 142:436-39, 1985.

5.

J. Parsonnet et al., Helicobacter pylori infection and the risk of gastric

carcinoma, N Engl J Med, 325:1127-31, 1991.

6.

S. Kado et al., Intestinal microflora are necessary for development of spontaneous

adenocarcinoma of the large intestine in T-cell receptor chain and p53 doubleknockout mice, Cancer Res, 61:2395-98, 2001.

7.

J.V. Newman et al., Bacterial infection promotes colon tumorigenesis in ApcMin/+

mice, J Infect Dis, 184:227-30, 2001.

8.

S.E. Erdman et al., CD4+ CD25+ regulatory T lymphocytes inhibit microbially

induced colon cancer in Rag2-deficient mice, Am J Pathol, 162:691-702, 2003.

9.

J.P. Zackular et al., The human gut microbiome as a screening tool for colorectal
cancer, Cancer Prev Res, 7:1112-21, 2014.

10. G. Zeller et al., Potential of fecal microbiota for early-stage detection of colorectal
cancer, Mol Syst Biol, 10:766, 2014.
11. J.P. Zackular et al., The gut microbiome modulates colon tumorigenesis, mBio,
4:e00692-13, 2013.
12. J.P. Zackular et al., Manipulation of the gut microbiota reveals role in colon
tumorigenesis,mSphere, doi:10.1128/mSphere.00001-15, 2015.
13. V.P. Rao et al., Innate immune inflammatory response against enteric bacteria
Helicobacter hepaticus induces mammary adenocarcinoma in mice, Cancer Res,
66:7395, 2006.
14. A. Sivan et al., Commensal Bifidobacterium promotes antitumor immunity and
facilitates anti-PD-L1 efficacy, Science, 350:1084-89, 2015.
15. M. Vtizou et al., Anticancer immunotherapy by CTLA-4 blockade relies on the gut
microbiota,Science, 350:1079-84, 2015.
..

Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells

Koji Atarashi Takeshi Tanoue Minoru Ando Nobuhiko Kamada .. Yoshinori Umesaki Kenya
Honda
ABSTRACT: Intestinal Th17 cells are induced and accumulate in response to colonization with a
subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain
extracellular pathogens. Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is
a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous
to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal
ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli
O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed
to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal
samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of
Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics.. Sep 2015
Cell 163(2)

https://www.researchgate.net/profile/Shoichi_Kado

http://dx.doi.org:/10.1016/j.cell.2015.08.058
MIT Faculty Newsletter

Vol. XXII No. 2 Nov / Dec 2009 Memorial Resolution for David B.

Schauer
We regretfully acknowledge the sudden death of our friend and colleague, Dr. David Schauer, on June
7, 2009 one day after his 48th birthday. David Schauer was known not only for his keen scientific
mind, but also as a friend whose empathy and compassion touched countless individuals.
Davids research was supported on a continuous basis by the National Institutes of Health throughout
his career at MIT. His studies on microbial pathogenesis of gastrointestinal pathogenic bacteria,
particularly Citrobacter rodentium, a murine model of enteropathogenic Escherichia coli, and
enterohepatic helicobacter are widely known and respected.
Davids research provided important insights into the molecular mechanisms evoked by enteric
pathogens and how the infections caused by these bacteria perturb the gastrointestinal barrier, elicit
inflammation, and produce clinically relevant disease.
Susan Erdman is a Principal Research Scientist and Assistant Director in the Division of Comparative
Medicine at MIT.

Invited Speaker. National Cancer Institute (NCI) Mucosal, Immunosurveillance,

Inflammation and Cancer Workshop. April, 2005.

Invited reviewer. National Cancer Institute (NCI) RFA-CA-06-014 Tumor

Microenvironment Network. August, 2006.

Invited speaker. Keystone Symposium on Mechanisms Linking Inflammation and

Cancer, February, 2007

Alumni Fellow, College of Veterinary Medicine, Mississippi State University, 2007

Invited Speaker. National Cancer Institute (NCI) Changing Concepts in Cancer Etiology:
The Role of the Human Microbiome. November, 2009.

Invited speaker. Keystone Symposium on Role of Inflammation in Oncogenesis,

February, 2010

Invited reviewer. National Cancer Institute (NCI) Tumor Microenvironment Network.

June, 2011

Invited Speaker, National Institute of Health (NIH) Human Microbiome Science

Conference: Vision for the Future. Wound healing to longevity: Microbe-induced
immune proficiency for human health. July, 2013.

Invited speaker, Wellcome Trust, Human Host-Microbiome Interactions Conference,

April 2014.

Invited speaker, Van Andel Institute, Origins of Cancer Conference, July 2014

Peer Bork

Current position: senior group leader (bioinformatics) at the EMBL (Heidelberg);

joint coordinator of the EMBL Structural and Computational Biology unit;
visiting group leader at the MDC (Berlin-Buch)

Advisor: Current and former member of various SABs and evaluation panels
(academia + industry)
Cofounder of LION bioscience AG (now SYGNIS Pharma AG), Cellzome AG,Anadys
Pharmaceuticals Inc. and biobyte solutions GmbH

Scientific interests: prediction of protein function at different spatial scales,

protein and genome evolution (iTOL), protein domain analysis,
comparative genome and proteome analysis, bridging genotype and phenotype

Some Projects: genome annotation,

comparative analysis of protein modules (SMART),
biochemical pathways and networks (STRING),
networks of chemicals and proteins (STITCH)
bioinformatics applications in molecular medicine
comparative metagenomics

List of group publications

Microbially Driven TLR5-Dependent Signaling Governs Distal Malignant Progression through

Tumor-Promoting Inflammation
Melanie R. Rutkowski, Tom L. Stephen, Nikolaos Svoronos, ., Julia Tchou, Gabriel A.
Rabinovich, Jose R. Conejo-Garcia
Cancer cell

12 Jan 2015; Volume 27, Issue 1, p2740

http://dx.doi.org/10.1016/j.ccell.2014.11.009

http://www.cell.com/cms/attachment/2023542033/2043769432/fx1.jpg

TLR5-dependent IL-6 mobilizes MDSCs that drive galectin-1 production by T cells

IL-17 drives malignant progression in IL-6-unresponsive tumors

TLR5-dependent differences in tumor growth are abrogated upon microbiota depletion

A common dominant TLR5 polymorphism influences the outcome of human cancers

The dominant TLR5R392X polymorphism abrogates flagellin responses in >7% of humans. We report that
TLR5-dependent commensal bacteria drive malignant progression at extramucosal locations by
increasing systemic IL-6, which drives mobilization of myeloid-derived suppressor cells (MDSCs).
Mechanistically, expanded granulocytic MDSCs cause lymphocytes in TLR5-responsive tumors to
secrete galectin-1, dampening antitumor immunity and accelerating malignant progression. In
contrast, IL-17 is consistently upregulated in TLR5-unresponsive tumor-bearing mice but only
accelerates malignant progression in IL-6-unresponsive tumors. Importantly, depletion of commensal
bacteria abrogates TLR5-dependent differences in tumor growth. Contrasting differences in
inflammatory cytokines and malignant evolution are recapitulated in TLR5-responsive/unresponsive
ovarian and breast cancer patients. Therefore, inflammation, antitumor immunity, and the clinical
outcome of cancer patients are influenced by a common TLR5 polymorphism.
see also Immune Influence

In recent years, research has demonstrated that microbes living in and on the mammalian body can
affect cancer risk, as well as responses to cancer treatment.
By Kate Yandell | April 1, 2016
http://www.the-scientist.com/?articles.view/articleNo/45644/title/Immune-Influence
Although the details of this microbe-cancer link remain unclear, investigators suspect that the
microbiomes ability to modulate inflammation and train immune cells to react to tumors is to blame.
Here are some of the hypotheses that have come out of recent research in rodents for how gut
bacteria shape immunity and influence cancer.

HOW THE MICROBIOME PROMOTES CANCER

Gut bacteria can dial up inflammation locally in the colon, as well as in other parts of the body, leading
to the release of reactive oxygen species, which damage cells and DNA, and of growth factors that
spur tumor growth and blood vessel formation.

http://www.the-scientist.com/images/April2016/ImmuneInfluence1_640px.jpg
http://www.the-scientist.com/images/April2016/ImmuneInfluence2_310px1.jpg
Helicobacter pylori can cause inflammation and high cell turnover in the stomach wall, which may lead
to cancerous growth.

HOW THE MICROBIOME STEMS CANCER

Some gut bacteria (e.g.,Bifidobacterium)
Gut bacteria can also produce factors that lower appear to activate dendritic cells,
inflammation and slow tumor growth.
which present cancer-cell antigens to T cells
that in turn kill the cancer cells.
http://www.the-scientist.com/images/April2016/ImmuneInfluence3_310px1.jpg
http://www.the-scientist.com/images/April2016/ImmuneInfluence4_310px1.jpg
Read the full story.

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Meeting Announcement: Cancer Immunotherapy and Combinations June

15-16 2016
Posted in cancer, Cancer and Therapeutics, FDA, FDA Regulatory
Affairs,Immunodiagnostics, immunology, immunotherapy, tagged bispecific antibodies, cancer,Cancer
immunology, cancer immunotherapeutics, Cancer immunotherapy, cancer vaccine,PD1, vaccine on February 12,
2016 | Leave a Comment

Rate This

Meeting Announcement: Cancer Immunotherapy and Combinations June 15-16

2016

Cancer Immunotherapy & Combinations June 15-16, 2016 in Boston, MA

#CHIWPC16

Final Brochure PDF | Learn More | Sponsorship & Exhibit Details | Register by March 4 & SAVE up
to $400!
Cambridge Healthtech Institutes inaugural Cancer Immunotherapy and Combinations meeting
will convene immuno-oncology researchers, cancer immunotherapy developers, and technology
providers to discuss next-generation approaches and combinations, including small molecule
development, to enhance the efficacy of checkpoint inhibitors.

BISPECIFIC ANTIBODIES DUAL TARGETING

FEATURED PRESENTATION: ANTI-PD1 OR CD137 ENHANCES NK-CELL
CYTOTOXICITY TOWARDS CD30+ HODGKIN LYMPHOMA INDUCED BY CD30/CD16A
TANDAB AFM13
Martin Treder, Ph.D., CSO, R&D, Affimed
In vivo Efficacy of Bispecific Antibodies Targeting Two Immune-Modulatory Receptors
Jacqueline Doody, Ph.D., Vice President, Immunology, F-star Biotechnology, Ltd
Dual-Targeting Bispecific Antibodies for Selective Neutralization of CD47 on Cancer Cells
Krzysztof Masternak, Ph.D., Head, Biology, Therapeutic Antibody Discovery, Novimmune

Update on MCLA-134: A Biclonics Binding Two Immunomodulatory Targets Expressed by T

Cells
Mark Throsby, Ph.D., CSO, Merus
The ImmTAC Technology: A Cutting-Edge Immunotherapy for Cancer Treatment
Samir Hassan, Ph.D., Director, Translational Research & Development, Immunocore Ltd.

RADIOTHERAPY AND CHEMOTHERAPY PD-1

COMBINATIONS
Rational Development of Combinations of Antiangiogenic Therapy with Immune Checkpoint
Blockers Using Mouse Models of HCC and Cirrhosis
Dan Duda, D.M.D., Ph.D., Associate Professor, Steele Laboratories for Tumor Biology, Department
of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School
Harnessing the Immune Microenvironment of Gastrointestinal Cancers Using Combined
Modalities
Osama Rahma, M.D., Assistant Professor, Internal Medicine/Oncology, University of Virginia

AGONIST PD-1 AND CTLA-4 COMBINATIONS

The Role of the Target in the Disposition and Immunogenicity of an Anti-GITR Agonist
Antibody
Enrique Escandn, Ph.D., Senior Principal Scientist, DMPK and Disposition, Merck
Combination of 4-1BB Agonist and PD-1 Antagonist Promotes Antitumor Effector/Memory
CD8 T Cells
Changyu Wang, Ph.D., Director, Cancer Immunology, Pfizer
Combination Immunotherapy with Checkpoint Blockade, Agonist Anti-OX40 mAb, and
Vaccination Rescues Anergic CD8 T Cells
William Redmond, Ph.D., Associate Member, Laboratory of Cancer Immunotherapy, Earle A. Chiles
Research Institute, Providence Portland Medical Center

Interactive Breakout Discussion Groups with Continental

Breakfast
This session features various discussion groups that are led by a moderator/s who ensures focused
conversations around the key issues listed. Attendees choose to join a specific group and the small,
informal setting facilitates sharing of ideas and active networking. Continental breakfast is available

for all participants.

Topic: Small Molecule Targeting of IDO1 and TDO for Cancer Immunotherapy
Moderator: Rogier Buijsman, Ph.D., Head, Chemistry, Netherlands Translational Research Center
B.V. (NTRC)
Overcoming challenges of current IDO1 inhibitors lacking selectivity over TDO and
having suboptimal drug-like properties
Advances in IDO1 and TDO inhibitor screening
Is selective IDO1 or TDO inhibitors is required, or a dual IDO1/TDO inhibitor is preferred
to obtain optimal efficacy and safety in the clinic?
Topic: Strategies for Developing Bispecific Antibodies for Cancer Immunotherapy
Moderator: Krzysztof Masternak, Ph.D., Head, Biology, Therapeutic Antibody Discovery,
Novimmune
Considerations for efficacy in vitro and in vivo: selectivity for tumor cells, ADCP,
ADCC, in vivoefficacy (xenograft models)
Insights into mechanisms of action
Safety considerations: binding selectivity, PK and tox studies
Topic: Combining Standard Antiangiogenic Therapy with Immune Checkpoint Inhibitors
Moderator: Dan Duda, D.M.D., Ph.D., Associate Professor, Steele Laboratories for Tumor Biology,
Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School
Will checkpoint combination with chemotherapy or other targeted agents prove to have too
many toxicity issues?
How do we minimize overlapping toxic effects of radiation and immunotherapy?
How to optimize the sequencing of these two treatment modalities?

SMALL MOLECULE INHIBITORS AS SINGLE AND

CHECKPOINT COMBINATION AGENTS
Selective Small Molecule Inhibitors of IDO1 and TDO for Cancer Immunotherapy
Rogier Buijsman, Ph.D., Head, Chemistry, Netherlands Translational Research Center B.V. (NTRC)
Potent and Selective Small Molecule USP7 Inhibitors for Cancer Immunotherapy
Suresh Kumar, Ph.D., Director, R&D, Progenra, Inc.
Epigenetic Agents for Combination with Cancer Immunotherapy
Svetlana Hamm, Ph.D., Head, Biology, Translational Pharmacology, 4SC Group

VACCINES AND CHECKPOINT BLOCKADE

IMMUNOTHERAPY
Immunotherapy for Mesothelioma with an in vivo DC Vaccine and PD-1/PD-L1 Blockade

Huabiao Chen, M.D., Ph.D., Principal Investigator, Vaccine and Immunotherapy Center,
Massachusetts General Hospital
Bringing Together Checkpoint Inhibition with Vaccines Using Customizing Capsids
Willie Quinn, Ph.D., President & CEO, Bullet Bio
Recommended All Access Package:
June 14 SC1: Immunosequencing: Generating a New Class of Cancer Immunotherapy Diagnostics*
June 14 SC5: Convergence of Immunotherapy and Epigenetics for Cancer Treatment*
June 14 SC8: Rational Design of Cancer Combination Therapies*
June 15-16: Cancer Immunotherapy and Combinations
June 16-17: Tumor Models for Cancer Immunotherapy
* Separate registration required.
Exhibit booth space has sold out! The few remaining spaces are being sold via sponsorship
only. To customize yoursponsorship package, please contact:
Joseph Vacca, M.Sc., Associate Director, Business Development, 781-9725431, jvacca@healthtech.com
For more information visit
WorldPreclinicalCongress.com/Cancer-Immunotherapy-Combinations
Cambridge Healthtech Institute, 250 First Avenue, Suite 300, Needham, MA
02494healthtech.com
Tel: 781-972-5400 | Fax: 781-972-5425
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Leaders in the CAR-T Field Are Proceeding With Cautious Hope

Posted in Cancer and Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy,Clinical &
Translational, Immuno-Oncology & Genomics, immunology, immunotherapy, tagged Adult ALL, autologous
transplant, B cell malignancies, cancer, cancer immunotherapeutics, Cancer immunotherapy, CART
therapy, Cellectis, childhood leukemia,follicular lymphoma, leukemia, novartis, University of Pennsylvania on
December 8, 2015 |Leave a Comment

Rate This

Leaders in the CAR-T Field Are Proceeding With Cautious Hope

Reporter: Stephen J. Williams, Ph.D.
It wasnt a long time ago, in fact the May 26, 2014 Cover Story in Forbes entitled Is This How Well
Cure Cancer with cover photo of Novartis CEO Joseph Jimenez and subtitle Will This man Cure
Cancer? highlighted the promise of CAR-T therapy as the magic bullet therapy which will eventually
cure all cancer. However, over the years, the pioneers of such therapy, while offering impressive
clinical results, caution not to get to eager in calling CAR-T as the end-all-be-all cure but insist there
are many issues that need be resolved.

The Allogenic Approach

In an interview for LabBiotech.eu Phillip Hemme had a discussion (and wonderful writeup) with Andr
Choulika, the CEO of the French CAR-T miracle Cellectis on the current state of CAR-T therapy for
cancer. Below is the interview in full as ther ae multiple important point Dr. Choulika mentioned,
including how much is needed to be done in the field.

Cellectis CEO: Im just trying to be realistic, CAR-T is not THE miracle cure for
Cancer

CAR-T is solidifying in everybodys mind as the next revolution in Cancer treatment. But
there is still a lot to doThats basically what came out from my discussion with Andr
Choulika, the CEO of the French CAR-T miracle Cellectis.
Cellectis is probably the most successful Biotech in France. It was founded in 1999 by Choulika himself
(not alone though), following the discovery of meganucleases ability to change gene editing. Today,

Cellectis is a well-known Biotech company counting over 100 employees end of October and having a
market cap north of 1 Billion euros.
The company is now focused on the development of allogenic CAR-T (from generic donors i.e. not
from the patient themself). With these universal CAR-T candidates (UCARTs). Cellectis has signed
a massive partnership with the French pharma Servier, as well as Pfizer (which owns 8% of Cellectis),
and has just announced two big milestones for the company within the last few weeks.
It is now able to produce its allogenic CAR-T in a GMP settings and it releases results from the
miracle treatment of a 11-month old girl from the UK with multi-resistant leukemia.

Lets start directly with the latest newsPeople seemed over-enthusiastic about UCART19
even the New York Times wrote about it. What do you think?
Its a great news for Cellectis even though its still a very early result, in a single patient only. Whats
important for us is that the first human patient received our treatment without showing any adverse
effects (such as no cytokinetic storm) and our CAR-T cells were still active in the body 3 months after
the injections.
Now, we have to expand the clinical trials to several patients and showing data from a cohort of
patients. We are now on track to file the clinical trial application by the end of the year.
Your approach in the CAR-T is pretty unique. You are using donors cells to treat many
different patients, whereas most CAR-T approaches are autologous (i.e. engineered the
patients own cells). Is the future in CAR-T the allogenic approach alone?
When we started to move into the CAR-T field we were pretty reluctant because there are not many
examples of commercial success in the field yet. But CAR-T has still attracted many big players such
as Novartis, Celgene, Juno or Kite. These each have a strong involvement in making autologous
therapies work commercially (Celgene especially, which makes most of its revenue
from groundbreaking and pricey cancer drugs).
On our side, we want to make this therapy accessible to a larger population and have good market
access at the end. We have already pretty good reason to think it could work out well for us. Well see
though
Comment: Reuters published a report a few weeks ago estimating the cost of autologous CAR-T could
be above $450K per treatment, which would make it economically not realistic for the healthcare
payers.

CAR-T seems to be extremely hype right now. At BIO-Europe 2015, I had the impression
everybody was talking about CAR-T. Do you think it could have the same impact as
monoclonal antibodies?
Whats interesting with CAR-T is that you can target cells which expresses less receptors (10k
receptors instead of 100k for monoclonal antibodies). This increases the targets for CAR-T and the
possibilities linked.
But there are also downsides. Tissues with low expressions can become targets too and CAR-T cells
would start attacking healthy cells.
People should not overemphasise CAR-T. We are still at the beginning of the beginning of this
technology. And it will probably have to be combined with surgery or checkpoint inhibitors.

You seem pessimistic about CAR-T?

I am just trying to be more realistic, even though I am super positive about the technology. It will
bring something really great to Haematology field, but is not a cure for Cancer. Its more of a longhaul race in the right direction as opposed to fast results, and we expect great things perhaps 20
years down the line as opposed to 2016.
But yes, it will probably not be the miracle product some people are talking about.
As for every early technology, there are many challenges associated with its development.
What are the main ones worth discussing?
I would say you have four main challenges
The administration of the cells will be challenging. We have to find way of injecting repeated doses of
the product (to ensure the therapy is fully effective seeing as CAR-T cells have a limited lifespan). This
is difficult because of immunogenicty against the therapy.
Secondly, combination will play an essential role too and checkpoint inhibitors should be involved.
The last two are linked to the targets.
As I mentioned before, CAR-T can be too sensitive and one way to control that would be to induce
logic gates where the cells would only act if a combination of receptors would be present. The last
challenge is to find other antigens.

Most of the CAR-T cells today target the same antigen: CD19+. We should find new antigens and
many companies are on the track, including us.

An anti-CD19 CAR-expressing T cell recognizing a CD19+ (Source: Kochenderfer et al., Nature

Reviews Clinical Oncology 10, 267-276, doi: 10.1038/nrclinonc.2013.46)

Autologous CART therapy

Dr. Carl June of University of Pennsylvania, who has helped pioneer the field of CAR-T therapy for
leukemia, has also been cautiously hopeful on the progress of the therapy. In his 2015 AACR National
Meeting address, he highlighted some achievements they had with CAR-T therapy in both hematologic
as well as solid tumors however it was stressed that there is much work to do with regards to
optimization of the system, characterization of new tumor antigens for diverse tumor types, as well as
the need to develop optimal treatment strategies to mitigate toxicities. Indeed many of the pioneers in
the field have been proactive in helping to develop pharmacovigilance, safety, and regulatory
strategies (highlighted in a post found here: NIH Considers Guidelines for CAR-T therapy: Report
from Recombinant DNA Advisory Committee and mitigating toxicities in a post Steroids,
Inflammation, and CAR-T Therapy) and much credit should be given to these researchers.
https://youtu.be/1sA_oz_1P5E

Cancer Research Institutes Breakthroughs in Cancer Immunotherapy Webinar Series are offered free
to the public and feature informative updates from leaders in cancer immunotherapy, followed by a
moderated Q&A. On June 10, 2013, Carl H. June, M.D., a specialist in T cell biology and lymphocyte
activation at the Perelman School of Medicine, University of Pennsylvania, discussed his
groundbreaking work that has led to remarkable remissions of advanced cancer. He focused on recent
and ongoing successes in developing treatments with T cells that have been genetically engineered to
target cancer. Called chimeric antigen receptor T cells (CAR T cells), these modified immune cells have
proven effective at eliminating cancer in some patients, and offer great hope for this emerging
strategy in cancer immunotherapy. For more information on this webinar, or to register for upcoming
webinars, please visit www.cancerresearch.org/webinars.
Below are reports from the 2015 American Society of Hematology Conference by Novartis
on results from CTL109 CART therapy trials. One trial is on response rate in B-cell lymphomas
and follicular cell lymphomas while the second report is ongoing trial results in childhood refractory
ALL, both conducted at University of Pennsylvania.

Novartis presents response rate data for CART therapy CTL019 in lymphoma
(Ref: Global Post, NASDAQ, PR Newswire)
posted on FirstWorldPharma.com December 6th, 2015
By: Matthew Dennis
Novartis announced Sunday data from an ongoing Phase IIa study demonstrating that the
experimental chimaeric antigen receptor T-cell (CART) therapy CTL019 led to an overall response rate
(ORR) at three months of 47 percent in adults with relapsed or refractory diffuse large B-cell
lymphoma (DLBCL) and an ORR of 73 percent in adults with follicular lymphoma. The results of the
trial were presented at the American Society of Hematology annual meeting.
Findings from the study, which was conducted by the University of Pennsylvanias Perelman School of
Medicine, include 15 adults with DLBCL and 11 with follicular lymphoma who were evaluable for
response. Results showed that three patients with DLBCL who achieved a partial response (PR) to
treatment at three months converted to complete response (CR) by six months. In addition, three
patients with follicular lymphoma who achieved a PR at three months converted to CR by six months.
Novartis added that one DLBCL patient with a PR at three months experienced disease progression at
six months after treatment. Further, one follicular lymphoma patient with a PR at three months who
remained in PR at nine months experienced disease progression at approximately 12 months after

treatment. The company indicated that median progression-free survival was 11.9 months for patients
with follicular lymphoma and 3 months for those with DLBCL.
In the study, four patients developed cytokine release syndrome (CRS) of grade 3 or higher. Novartis
noted that during CRS, patients typically experience varying degrees of influenza-like symptoms with
high fevers, nausea, muscle pain, and in some cases, low blood pressure and breathing difficulties.
Meanwhile, neurologic toxicity occurred in two patients in the trial, including one grade three episode
of delirium and one possibly related grade five encephalopathy.
These data add to the growing body of clinical evidence on CTL019 and illustrate its potential benefit
in the treatment of relapsed and refractory non-Hodgkin lymphoma, commented lead investigator
Stephen Schuster. Novartis indicated that the findings keep CTL019 on track for submission to the FDA
in 2017. Usman Azam, global head of Novartis cell and gene therapies unit, said we remain
consistent again with the data set.
Its an attractive population, its a population that continues to have a huge unmet need, its a
cornerstone of our investments, Azam remarked. Analysts expect CART therapies, once approved, to
cost up to $450 000 per patient. Novartis acknowledged that prices will be high, but declined to give
further details. With any disruptive innovation that comes, initially, cost of goods is very challenging,
Azam said, adding as time goes on, and more patients are treated, we will simplify that cost base.
Source: http://www.firstwordpharma.com/node/1338217?tsid=28&region_id=2#axzz3tfDVaT1f

Novartis AG (NVS)s Experimental Therapy Wipes Out Blood Cancer in 93 Percent of Patients
Reported in Biospace.com (for full article see here)
Novaritis and University of Pennsylvania reported results of the CTL019 CART trials for the treatment
of children with relapsed/refractory acute lymphoblastic leukemia at the 2015 Annual Hemotologic
Society Meeting. 55 of 59 patients, or 93 percent, experienced complete remissions with CTL019. The
study did show that at the end of one year, 55 percent of patients had a remission-free survival rate
and that 18 patients continued to show complete remission following one year

Other posts on the Open Access Journal on CAR-T therapy include

CAR-T therapy in leukemia

Steroids, Inflammation, and CAR-T Therapy
NIH Considers Guidelines for CAR-T therapy: Report from Recombinant
DNA Advisory Committee

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Cancer Immunotherapy
Posted in Cancer and Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy,Cancer Informatics, Clinical
& Translational, Clinical Diagnostic, Computational Biology/Systems and Bioinformatics, Curation, Disease
Biology, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genome Biology, Human Immune
System in Health and in Disease, Immuno-Oncology & Genomics, immunology, tagged Cancer
immunotherapy, immune tolerance, PDL1/PD1 pathway on October 27, 2015 | 1 Comment

1 Vote

Cancer Immunotherapy
Curator: Larry H. Bernstein, MD, FCAP, Curator

Research explains limits of cancer immunotherapy drugs

Finding offers possibility to improve response to therapies
http://www.uofmhealth.org/news/archive/201510/research-explains-limits-cancer-immunotherapydrugs

A new study from the University of Michigan Comprehensive Cancer Center reveals molecular changes
within the tumor that are preventing the immunotherapy drugs from killing off the cancer.

Clinical trials with PD-L1 and PD-1 blockade suggested that tumors with a high number of
inflammation-causing T cells were more responsive to the immunotherapy-based PD-L1 and PD-1
inhibitors. Tumors with low inflammation, or low T cells, were less responsive. But what controls T
cells in the tumor microenvironment is poorly understood.

http://www.uofmhealth.org/sites/default/files/styles/large/public/Immunotherapy.png?itok=_oihl0jj

We defined a molecular mechanism to explain why some tumors are inflamed and others are not
and consequently why some patients will be responsive to therapy and others not, says senior
author Weiping Zou, M.D., Ph.D.
If we can reprogram this epigenetic mechanism, then the therapy might work for more patients,
says Zou, Charles B. de Nancrede Professor of Surgery, Immunology and Biology at the University of
Michigan Medical School.
Zous group was the first to show PD-L1 expression, regulation and functional blockade in dendritic
cells in the human cancer microenvironment.
In this study, published in Nature, researchers studied human and mouse models of ovarian
cancer cells. They applied epigenetic drugs and found that the numbers of T cells in the tumor
increased. They also saw that the epigenetic drugs synergized the anti-tumor effect of PD-L1 blockade
in their models.
We hope this could be developed into a clinical trial testing a combination of PD-L1 and PD-1 blockade
with epigenetic therapy. We want to see if we can make the responders more responsive and turn the
non-responders into responders, Zou says.

Additional authors: From U-M: Dongjun Pen, Ilona Kryczek, Nisha Nagarsheth, Lili Zhao, Shuang
Wei, Weimin Wang, Yuqing Sun, Ende Zhao, Linda Vatan, Wojciech Szeliga, Yali Dou, Kathleen Cho,
Rebecca Liu; from Medical University in Lublin, Poland: Jan Kotarski, Rafal Tarkowski; from Henry Ford
Health System: Sharon Hensley-Alford, Adnan Munkarah
Funding: National Institutes of Health grants CA190176, CA123088, CA099985, CA193136,
CA152470, CA171306, 5P30 CA46592; Rivkin Ovarian Cancer Center, Ovarian Cancer Research Fund,
Barbara and Don Leclair
Cancer immunotherapy research is evolving to more targeted strategies
Discoveries in immune pathway research have helped refine cancer immunotherapy strategies to
become more targeted.1,2

http://www.researchcancerimmunotherapy.com/overview/targeted-cancer-research

CAR=chimeric antigen receptor;

CSF-1R=colony-stimulating factor 1; CTLA4=cytotoxic

T-lymphocyte antigen-4; IDO=indoleamine

2,3-dioxygenase; PD-1=programmed death-1; PD-L1=programmed death-ligand 1.

http://www.researchcancerimmunotherapy.com/images/overview/evolution-research/history-ofimmunotherapy.png

Engaging the immune response: a unique approach to cancer management

Cancer immunotherapy strategies are designed to engage the immune system against tumors. This
approach is unique in the oncology setting and introduces new considerations for cancer
management.1,2

TARGETED
to tumor-specific antigens

RAPID
activation of the immune response

ADAPTABLE
as the tumor mutates and evolves

SELF-PROPAGATING
with each revolution of the cancer immunity cycle

DURABLE
response over time

CONSIDERATIONS FOR CANCER IMMUNOLOGY

Duration of response
The immune response has the ability to adapt with cancer as it evolves, and can become selfpropagating once the cancer immunity cycle is initiated. Immune-directed strategies aim to leverage
these attributes, with the goal of inducing a durable antitumor effect. 3-5

Pseudo-progression
T-cell infiltration to the tumor site may cause an apparent increase in tumor size or the appearance of
new lesions. This inflammatory effect can be misinterpreted as progressive disease, as it can be
difficult to differentiate the different cell types in radiographic imaging. New criteria have been
developed to better capture immune-related response patterns, and may guide evaluation of
immunotherapies in clinical trials, and potentially in clinical care. 1,2,6

REFERENCES
1.

Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity

cycle. Immunity. 2013;39:1-10. PMID: 23890059

2.

Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of

age. Nature. 2011;480:480-489. PMID: 22193102

3.

Hanahan D, Weinberg RA. Hallmarks of cancer: the next

generation. Cell. 2011;144:646-674. PMID: 21376230

Immune-related adverse events

While the goal of cancer immunotherapy research is to understand how to activate specific
components of the immune response, the potential for off-target effects exists. Adverse event profiles

may vary among different immune-directed strategies. As strategies grow more targeted, the
recognition and management of immune-related adverse events will evolve. 1,3
EVADING IMMUNE DESTRUCTION
EVOLUTION OF RESEARCH

REFERENCES
1.

Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of

age. Nature. 2011;480:480-489. PMID: 22193102

2.

Hoos A, Eggermont AM, Janetzki S, et al. Improved endpoints for cancer

immunotherapy trials. J Natl Cancer Inst. 2010;102:1388-1397.PMID: 20826737

3.

Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity

cycle. Immunity. 2013;39:1-10. PMID: 23890059

4.

Topalian SL, Weiner GJ, Pardoll DM. Cancer immunotherapy comes of age. J Clin
Oncol. 2011;29:4828-4836. PMID: 22042955

5.

Chen DS, Irving BA, Hodi FS. Molecular pathways: next-generation immunotherapy
inhibiting programmed death-ligand 1 and programmed death-1. Clin Cancer Res.
2012;18:6580-6587.PMID: 23087408

6.

Wolchok JD, Hoos A, ODay S, et al. Guidelines for the evaluation of immune therapy
activity in solid tumors: immune-related response criteria. Clin Cancer
Res. 2009;15:7412-7420. PMID: 19934295

Tumors can evade immune destruction

By disrupting the processes of the cancer immunity cycle throughout the body, tumors can avoid
detection by the immune system and limit the extent of immune destruction. 1-3

Tumor microenvironment
Disrupting antigen detection

http://www.researchcancerimmunotherapy.com/images/overview/evading-immune-destruction/tumormicroenv.png

Lymph node
Inhibiting T-cell activation by dendritic cells
http://www.researchcancerimmunotherapy.com/images/overview/evading-immunedestruction/lymph-node.png

Blood vessel
Blocking T-cell infiltration into tumor
http://www.researchcancerimmunotherapy.com/images/overview/evading-immune-destruction/bloodvessel.png

Tumor microenvironment
Suppressing cytotoxic T-cell activity
http://www.researchcancerimmunotherapy.com/images/overview/evading-immunedestruction/suppressing-tcell.png

EVADING IMMUNE DESTRUCTION IS AN EMERGING HALLMARK OF CANCER

http://www.researchcancerimmunotherapy.com/images/overview/evading-immunedestruction/hallmark-cancer.png
The growing body of research into the mechanisms of immune evasion has led to its addition as an
emerging hallmark of cancer, a distinct biological capability that enables tumors to grow and
metastasize.3

MUTATION RATE BY CANCER TYPE4

http://www.researchcancerimmunotherapy.com/images/tumor-types/immunogeniccancer/immunogenic-cancer.png
REFERENCES
1.

Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity

cycle. Immunity. 2013;39:1-10. PMID: 23890059

2.

Chen DS, Irving BA, Hodi FS. Molecular pathways: next-generation immunotherapy
inhibiting programmed death-ligand 1 and programmed death-1. Clin Cancer Res.
2012;18:6580-6587.PMID: 23087408

3.

Rajasagi M, Shokla SA, Fritsch EF, et al. Systematic identification of personal tumorspecific neoantigens in chronic lymphocytic leukemia.Blood. 2014;124:453462. PMID: 24891321

4.

Lawrence MS, Stojanov P, Polak P, et al. Mutational heterogeneity in cancer and the
search for new cancer-associated genes.Nature.2013;499:214-218. PMID:
23770567

Atezolizumab (Anti-PDL1) clinical trials

Atezolizumab clinical trials are being conducted for the following types of cancers: lung, bladder,
kidney, hematological malignancies, and breast cancer. Please check back for updates or search
clinicaltrials.gov with the search term Atezolizumab.

EXPLORING A MORE PERSONALIZED APPROACH TO CANCER IMMUNOTHERAPY RESEARCH

With the evolution to more targeted strategies, research is focusing on identifying predictors of
individual immune response through specific tumor characteristics and factors in the tumor
microenvironment, such as

The presence of tumor-infiltrating immune cells 8

o

The ability of immune cells to infiltrate the tumor microenvironment may be

a key criterion for a variety of immune-directed strategies, and could
indicate which tumors are more likely to respond

Gene expression patterns in tumors, particularly the genes involved in immune

response9

Cell surface protein expression

o

PD-L1 expression on tumor cells and tumor-infiltrating immune cells 10,11

MUC1 expression on tumor cells 12

REFERENCES
1.

Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity

cycle. Immunity. 2013;39:1-10. PMID: 23890059

2.

Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of

age. Nature. 2011;480:480-489. PMID: 22193102

3.

Lesterhuis WJ, Haanen JB, Punt CJ. Cancer immunotherapyrevisited. Nat Rev Drug
Discov. 2011;10:591-600. PMID: 21804596

4.

National Institutes of Health

ClinicalTrials.gov.https://clinicaltrials.gov/ct2/show/NCT01494688. Accessed March
4, 2015.

5.

National Institutes of Health

ClinicalTrials.gov.https://clinicaltrials.gov/ct2/show/NCT00739609. Accessed March
4, 2015.

6.

Glienke W, Esser R, Priesner C, et al. Advantages and applications of CAR-expressing

natural killer cells. Front Pharmacol. 2015;6:21. doi:
10.3389/fphar.2015.00021. PMID: 25729364

7.

National Institutes of Health

ClinicalTrials.gov.https://clinicaltrials.gov/ct2/show/NCT01303705. Accessed March
4, 2015.

8.

Gajewski TF, Schreiber H, Fu YX. Innate and adaptive immune cells in the tumor
microenvironment. Nat Immunol. 2013;14:1014-1022.PMID: 24048123

9.

Ji RR, Chasalow SD, Wang L, et al. An immune-active tumor microenvironment

favors clinical response to ipilimumab. Cancer Immunol
Immunother. 2012;61:1019-1031. PMID: 22146893

10. Taube JM, Anders RA, Young GD, et al. Colocalization of inflammatory response with
B7-h1 expression in human melanocytic lesions supports an adaptive resistance
mechanism of immune escape. Sci Transl Med. 2012;4:127ra37. PMID: 22461641

11. Chen DS, Irving BA, Hodi FS. Molecular pathways: next-generation immunotherapy
inhibiting programmed death-ligand 1 and programmed death-1. Clin Cancer Res.
2012;18:6580-6587.PMID: 23087408
12. Stojnev S, Ristic-Petrovic A, Velickovic LJ, et al. Prognostic significance of mucin
expression in urothelial bladder cancer. Int J Clin Exp Pathol. 2014;7:49454958. PMID: 25197366
13. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev
Cancer. 2012;12:252-264. PMID: 22437870
14. Taylor RC, Patel A, Panageas KS, Busam KJ, Brady MS. Tumor-infiltrating
lymphocytes predict sentinel lymph node positivity in patients with cutaneous
melanoma. J Clin Oncol. 2007;25:869-875. PMID: 17327608

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Monoclonal Antibody Therapy and Market

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Genome Biology,Personalized and Precision Medicine
& Genomic Research, Translational Science, taggedcancer, Cancer immunology, Cancer immunotherapy, Demet
Sag, Monoclonal antibodieson October 14, 2015 | Leave a Comment

1 Vote

Monoclonal Antibody Therapy and Market

Demet Sag, PhD, CRA, GCP

Monoclonal Antibody treatment means a biological therapy where monoclonal antibodies is used to
initiate development of specific antibodies (protein molecules produced by the B cells as a primary
immune defense), so that they can fight against antigens (substances that are capable of inducing a
specific immune response) specifically to kill extracellular/ cell surface target. Thus, the application of
this types of therapies are not limited to cancer but also rheumatoid arthritis, multiple
sclerosis, Alzheimers disease, and some infectious diseases such as Ebola.

To eliminate or reduce the effects of chemotherapeutic agents. Thus chemotherapeutics agents

attached to monoclonal antibodies.

Diagnostic process:
Monoclonal antibodies again used as a vehicle to locate the tumorigenic cancer cells in the body. There
can be several methods but one of them is carrying radioactive substances to cancer cells so that they
can be labelled in vivo. However, there are less invasive ways to do as well. As a result, there are new
combination of methods such as:

nuclear imaging,

surgical mapping, and

direct therapy in multiple settings either alone, or in conjunction with

chemotherapeutic agents, adjuvant.

How do monoclonal antibody drugs work?

1.

Naked monoclonal antibodies:

Make the cancer cell more visible to the immune system.

Action is to boost immune system.

Example: Alemtuzumab (Campath), chronic lymphocytic leukemia (CLL) by binding to the CD52
antigen on lymphocytes.

Block immune checkpoint inhibitor proteins

Treatments that target PD-1 or PD-L1.

PD-1 is a checkpoint protein on T cells, called off switch of T cells since PD-1 prevents from
attacking other cells in the body. Yet, when it is overexpressed on the cancer cells, tumors escape
from immune system, because when PD-1 binds to PD-L1, T cells thinks these cells are bodys own
normal cells.
http://www.nature.com/nature/journal/v515/n7528/images/515496a-f1.jpg

a, Tumour cells express both cancer-driving mutations and passenger mutations that cause the
expression of neoantigens new molecular structures that, when presented by MHC proteins on the
cell surface, are recognized by T cells of the immune system as being foreign, leading to an immune
response against the tumour. However, interactions between the receptor PD-1 and its ligand PD-L1,
which are expressed on tumour cells, T cells and other immune cells such as macrophages, activate
signalling pathways that inhibit T-cell activity and thus inhibit the antitumour immune response. b,
Antibodies that block the PD-1 pathway by binding to PD-1 or PD-L1 can reactivate T-cell activity and
proliferation, leading to enhanced antitumour immunity.

Examples are:

Pembrolizumab (Keytruda)

Nivolumab (Opdivo)

There is a possibility of developing an autoimmune reaction. The most common side effects include
fatigue, cough, nausea, skin rash, and itching. Rarely more serious problems in the lungs, intestines,
liver, kidneys, hormone-making glands, or other organs may occur.
Treatments that target CTLA-4
Another protein is CTLA-4 to control T cells, off switch.

Example: Ipilimumab (Yervoy)is a monoclonal antibody that attaches to CTLA-4 and stops it from
working. This can boost the bodys immune response against cancer cells.

Block antigens on cancer cells (or other nearby cells).

Example: Trastuzumab, when HER2 is activated, binds to these proteins and stops antigens from
becoming active in breast and stomach cancer cells.
Example: Rituxan specifically attaches to CD20 that is found only on B cells so when these labelled B
cells can be visible to immune system. There are certain types of lymphomas predisposed due to
malfunctioning B cells.

Block growth signals. Prevent signal amplification for cell growth.

The cells like to amplify their message in danger or during certain metabolisms so they secrete or
produce a type of chemicals called growth factors. These factors then attaches to specific receptors
on the surface of normal cells and cancer cells. Thus, signaling the cells to grow faster than the normal
cells. The action is preventing the signals to be received by monoclonal.
Example:

Cetuximab (Erbitux), targets epidermal growth factor. Thus its function utilized to cure colon cancer,
head and neck cancers.

Stop new blood vessels from forming.

Tumors needs to grow so in the body they need blood vessel formation to feed the cell growth
(angiogenesis)
Example; Bevacizumab (Avastin) targets vascular endothelial growth factor (VEGF) and blocks the
angiogenesis.

2.

Conjugated monoclonal antibodies (tagged, labeled, or loadedantibodies).

Deliver chemotherapy to cancer cells.

They are monoclonal antibodies (mAbs) joined to a chemotherapy drug or to a radioactive particle to
locate cancer cells directly through targeting specific antigen after circulating in the bloodstream. They
are used as a homing device.
Chemo-labeled antibodies: Also called as antibody-drug conjugates (ADCs) and provide powerful
chemotherapy (or other) drugs attached to them.

Brentuximab vedotin (Adcetris), an antibody that targets the CD30 antigen on

lymphocytes, attached to MMAE (a chemo drug) againstHodgkin lymphoma and
anaplastic large cell lymphoma.

Ado-trastuzumab emtansine (Kadcyla, also called TDM-1), an antibody that

targets the HER2 protein, attached to DM1 (a chemo drug) against cells
overexpressing HER2 in breast cancer

Toxin attached protein: Denileukin diftitox (Ontak ) is not an antibody but it is a protein,
cytokine known as interleukin-2 (IL-2) and attached to diphtheria toxin that recognizes CD25 antigen
to treat lymphoma of the skin (cutaneous T-cell lymphoma).

Radiolabeled antibodies: Deliver radiation to cancer cells.

The other method, less preferred, is radiation-linked monoclonal antibodies. This time low radiation in
long term used to target the cancer cells but it is suggested that this method has elevated outcome to
kill the cancer cells than conventional high-dose external beam radiation.

Example; Ibritumomab (Zevalin), is an approved treatment. The targeted disease is for non-Hodgkins
lymphoma.
Treatment with this type of antibody also referred as radioimmunotherapy (RIT).

3.

Bispecific monoclonal antibodies

If the drug contains two parts of 2 different mAbs, meaning they can attach to 2 different proteins at
the same time, they are called Bispecific monoclonal antibodies since they attack two proteins
at the same time.

Example: Blinatumomab (Blincyto), can attach CD 19 which is found on some leukemia and
lymphoma cells and CD3 on T cells. Thus, brings opponents, immune and malignant cancer cells, to
defeat cancer.

THE OTHER SIDE OF THE COIN: SAFETY

Possible side effects of monoclonal antibodies
Delivery is intravenously and since Mabs are themselves are proteins sometimes presents side effects
like an allergic reaction yet compared to chemotherapy drugs these effects are much less. .

Fever

Chills

Weakness

Headache

Nausea

Vomiting

Diarrhea

Low blood pressure

Rashes

Examples:

Bevacizumab (Avastin), high blood pressure, bleeding, poor wound healing, blood
clots, and kidney damage.

Cetuximab (Erbitux), serious rashes in some people.

Manufacturing of Monoclonal Antibodies and Market

Since 2000, the therapeutic market for monoclonal antibodies has grown exponentially. The current
big 5 therapeutic antibodies on the market arebevacizumab, trastuzumab (both
oncology), adalimumab, infliximab (bothautoimmune and inflammatory disorders, AIID)
and rituximab (oncology and AIID) accounted for 80% of revenues in 2006. In 2007, eight of the 20
best-selling biotechnology drugs in the U.S. are therapeutic monoclonal antibodies. Scolnik, Pablo A.
(2009). mAbs: A business perspective. MAbs 1 (2): 179
184.doi:10.4161/mabs.1.2.7736. PMC 2725420. PMID 20061824.
This rapid growth in demand for monoclonal antibody production has been well accommodated by the
industrialization of mAb manufacturing. Kelley, Brian (2009). Industrialization of mAb production
technology. MAbs 1 (5): 443452.doi:10.4161/mabs.1.5.9448. PMC 2759494. PMID 20065641.

http://www.ncbi.nlm.nih.gov/pmc/art
icles/PMC2759494/bin/mabs0105_0443_fig001.jpg
Model mAb production plant design and capabilities. A model large scale mAb production plant
employs multiple bioreactors configured to supply a single purification train. A plant having six
individual 15 kL bioreactors is potentially capable of supplying 10 tons of purified mAb per year using
conventional technologies, or 45 products with 1 ton demands. This enormous capacity per plant
would result in a marked decrease in drug substance production costs, and results in significant excess
capacity throughout the biopharmaceutical industry.
Production:
Production capacity estimates for mammalian cell-derived mAbsa

Year
2007
2010
2013
a

CMO
500 kL
700 kL
1,000 kL

Product company
1,800 kL
2,700 kL
3,000 kL

Total
2,300 kL
3,400 kL
4,000 kL

Capacity at 2 g/L
70 tons/yr
100 tons/yr
120 tons/yr

Capacity at 5 g/L
170 tons/yr
255 tons/yr
300 tons/yr

Capacity estimates from ref. Ransohoff TC, Ecker DM, Levine HL, Miller J. Cell culture manufacturing

capacity: trends and outlook through 2013. PharmSource. 2008

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759494/bin/mabs0105_0443_fig003.jpg

Estimated demand for therapeutic mAbs and Fc-fusion products in 2009. The total demand for the top
15 mAbs and Fc-fusions in 2009 is estimated to be approximately 7 tons, with the four largest volume
products requiring approximately one ton per year. More than half of the products were estimated to
require less than 200 kg per year.

Distribution of average wholesale prices for mAb and Fc-fusions in 2008. The average U.S. wholesale
prices per gram for 15 commercial mAbs and Fc-fusions are shown. The minimum is approximately
$2,000 per gram, and the median is approximately $8,000 per gram. Note that a significant price
erosion (50% of the minimum shown here) for a product with modest demand (100 kg/yr) could
result in an unprofitable market, as revenues for the therapeutic product ($100 million/yr) may never
provide a positive return on investment.
Sensitivity analysis of mAb drug substance COGs for the model plant (six 15kL bioreactors)

Plant
Titer
capacity
(g/L)
(tons/yr)
0.5
2
5
a

1
4
10

Raw materials ($/gm)

Depreciation &
labor ($/gm)b

Cell
Purification
culturea
20
100
4
4
25
2
10

Fill/Finish
costs per vial
($)

Total Drug Product

Cost ($/vial)
100 mg
22
13
12

10

1
gm
134
43
26

Assumes medium cost of $8/L.

Based on the model plant ($500 M capital investment + 250 staff = $100 M per year).

Estimated cost breakdown for three production scenarios

Model largescale plant

Basis: 5 g/L
6 15 kL
a
Capital Investment $500 M
Depreciationb($/yr) $50 M
Raw Materialsc
$10/gm
Labor ($/yr)d
$50 M

Small-scale plant
using disposables
n 2 kL
$125 M
$12.5 M
$20/gm
$20 M

CMO

COGs
$/gm

10 ton/yr20
1 ton/yr 110
0.1
1,010

23
53
345

CMO
15 kL

$10/gm

$3
M/batche
60
60
60

Difference in annual cost for

two best alternatives ($M/yr)
$30 M
$7 M
$29 M

Model large- Small-scale plant

scale plant using disposables

CMO

ton/yr
a

The new facility based on disposables is assumed to cost just one-quarter of model plant to build, and

uses only the number of bioreactors (n) needed to satisfy the demand.
b

A 10-year straight line depreciation is used to estimate the depreciation costs.

Raw material costs per gram are assumed to be slightly higher for the disposable facility.

Labor costs for the new facility are assumed to be just 40% of the model plant (100 vs 250 staff,

respectively).
e

A constant cost per batch is assumed for the CMO, all-inclusive of production, testing and release.

Sales and Marketing

PMC full text: MAbs. 2009 Mar-Apr; 1(2): 179184.

FDA-approved marketed mAbs

Name
Generic

Structure Target Indication

Trade

Approval
Path
Sales
(Y)

Landing Expansion
(U.S.
$B)

First Tier
infliximab

%
Top
20

Remicade Ch

TNF

CD

RA

O, A,
4.6
P, F

$5.0

9.84

PP
O, P 5.1

$4.9

9.62

F, P 7.5
F, P 7.1

$4.3
$3.6

8.45
7.15

3.7

$3.1

6.04

A, P 9.7
P
6.8

$1.4
$1.2

2.73
2.39

AS
PA
UC
rituximab

Rituxan, Ch
MabThera

trastuzumab
bevacizumab

Herceptin Hm
Avastin
Hm

adalimumab

Humira

Hu

cetuximab
ranibizumab

Erbitux
Lucentis

Ch
Hm

CD20

NHL

RA
DLBC
1-NHL
HER2 mBC BC
VEGF mCRC mCRC
NSCLC
HER2BCa
TNF
RA
RA
JIA
PA
AS
CD
PP
EGFR mCRC SCCHN
VEGF AMD

Name
Generic
palivizumab

Structure Target Indication

Approval
Path
Sales
(Y)

%
Top
20

Trade
Synagis

Hm

RSV

Landing Expansion
RSV
P

tositumomab

Bexxar

Mu

CD20

NHLb

alemtuzumab

Campath

Hm

CD52

B-CLL B-CLLd

Cimzia

Hm

TNF

CD

Mylotarg

Hm

CD33

AML

P, A,
6.5
O

$60.0 0.12

CD3

OR

n/a

$150.0 0.30

CD11a
GP
IIb/IIIa
CD25
C5
a-4
integrin

PS

10e

$163.0 0.32

n/a

$380.0 0.75

O, P n/a
O, P n/a

$300.0 0.59
$230.0 0.45

3.6

Second Tier

certolizumab
pegol
gemtuzumab
ozogamicin
muromonabCD3
efalizumab

Orthoclone
Mu
Okt3
Raptiva Hm

abciximab

ReoPro

Ch

basiliximab
eculizumab

Simulect
Soliris

Ch
Hm

natalizumab

Tysabri

Hm

AC

NHLc

OR
CI

OR
PNH
MS

panitumumab Vectibix

Hu

EGFR mCRC

omalizumab
daclizumab
ibritumomab
tiuxetan

Xolair
Zenapax

Hm
Hm

IgE
CD25

AA
OR

Zevalin

Mu

CD20

NHL

13.7 $10.3
A, P,
10.4e
F

CD

ORp

n/a

10.6e

A, P,
7.4
F
9.7
O, P n/a
P, A,
10.2
O, F

$1.1 2.25
(U.S.
$M)
0.02
$108.0 0.21
n/a

n/a

$100.0 0.20
$365.0 0.72
$472.0 0.93
$60.0 0.12
$17.0 0.03

Abbreviations: Structure: Ch, chimeric; Hm, humanized; Hu, human; Mu, murine. Regulatory Path: A,
accelerated approval; F, fast-track; P, priority review; O, orphan indication. 1-, first-line therapy; a,
conditional approval; b, rituximab refractory; c, refractory to chemotherapy; d, single-agent; e,
estimate; m, metastatic; n/a, information not available; p, prophylaxis. Sources: 20 Compounds that
defined biotech, Signals online magazine at www.signalsmag.com; ReCap database;
Biopharmaceutical Products in the U.S. and European markets 6 th edition, Ronald A. Rader, ed; Pharma
Sales and BioPharmInsights databases; Reichert JM, Ph. D.; personal communications. Development
times and sales estimates for some Second Tier mAbs are based on limited information.
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Humanized Mice May Revolutionize Cancer Drug Discovery

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Humanized Mice May Revolutionize

Cancer Drug Discovery
Curator: Stephen J. Williams, Ph.D.
Decades ago cancer research and the process of oncology drug discovery was revolutionized by the
development of mice deficient in their immune system, allowing for the successful implantation of
human-derived tumors. The ability to implant human tumors without rejection allowed researchers to

study how the kinetics of human tumor growth in its three-dimensional environment, evaluate
potential human oncogenes and drivers of oncogenesis, and evaluate potential chemotherapeutic
therapies. Indeed, the standard preclinical test for antitumor activity has involved the subcutaneous
xenograft model in immunocompromised (SCID or nude athymic) mice. More detail is given in the
follow posts in which I describe some early pioneers in this work as well as the development of large
animal SCID models:
Heroes in Medical Research: Developing Models for Cancer Research
The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research
The SCID Pig II: Researchers Develop Another SCID Pig, And Another Great Model For
Cancer Research
This strategy (putting human tumor cells into immunocompromised mice and testing therapeutic
genes and /or compounds) has worked extremely well for most cytotoxic chemotherapeutics (those
chemotherapeutic drugs with mechanisms of action related to cell kill, vital cell functions, and cell
cycle). For example the NCI 60 panel of human tumor cell lines has proved predictive for the
chemosensitivity of a wide range of compounds.

The NCI-60 panel has been used by the Developmental Therapeutics Program (DTP) of
the U.S. National Cancer Institute (NCI) to screen >100,000 chemically defined
compounds plus a large number of natural product extracts for anticancer activity since
1990 [1-3]

Expression (microarray data) with bioinformatic strategies can use efficacy in one cell
type to predict efficacy in cell types not in the panel in A strategy for predicting the
chemosensitivity of human cancers and its application to drug discovery however in
Coombes, Wang and Baggerly in Genomic Signatures on the NCI60 cell lines DO
NOT predict patient response to chemotherapy, and [5] where the authors could not
reproduce the results in [4] and discussed in Putting Oncology Patients at Risk and
aRETRACTED paper in [6] relying on miRNA signatures.

Even though the immunocompromised model has contributed greatly to the chemotherapeutic drug
discovery process. using these models to develop the new line of immuno-oncology products has been
met with challenges three which I highlight below with curated database of references and examples.
From a practical standpoint development of a mouse which can act as a recipient for human
tumors yet have a humanized immune system allows for the preclinical evaluation of
antitumoral effect of therapeutic antibodies without the need to use neutralizing antibodies
to the comparable mouse epitope,

thereby reducing the complexity of the study and

preventing complications related to pharmacokinetics.

Champions Oncology Files Patents for Use of PDX Platform in ImmuneOncology

Hackensack, NJ August 17, 2015 Champions Oncology, Inc. (OTC: CSBR), engaged in the
development of advanced technology solutions and services to personalize the development and use of
oncology drugs, today announced that it has filed two patent applications with the United States
Patent and Trademark Office (USPTO) relating to the development and use of mice with humanized
immune systems to test immune-oncology drugs and therapeutic cancer vaccines.
Dr. David Sidransky, the founder and Chairman of Champions Oncology commented, Drug
development in the immune-oncology space is fundamentally changing our approach to cancer
treatment. These patents represent potentially invaluable tools for developing and personalizing
immune therapy based on cutting edge sequence analysis, bioinformatics and our unique in vivo
models.
Joel Ackerman, Chief Executive Officer of Champions Oncology stated, Developing intellectual
property related to our Champions TumorGraft platform has been an important component of
strategy. The filing of these patents is an important milestone in leveraging our research and
development investment to expand our platform and create proprietary tools for use by our
pharmaceutical partners. We continue to look for additional revenue streams to supplement our feefor-service business and we believe these patents will help us capture more of the value we create for
our customers in the future.
The first patent filing covers the methodology used by the Company to create a mouse model,
containing a humanized immune system and a human tumor xenograft, which is capable of testing the
efficacy of immune-oncology agents, both as single agents and in combination with anti-neoplastic
drugs. The second patent filing relates to the detection of neoantigens and their role in the
development of anti-cancer vaccines.
Keren Pez, Chief Scientific Officer, explained, In the last few years, there has been a significant
increase in cancer research that focuses on exploring the power of the human immune system to
attack tumors. However, its challenging to test immune-oncology agents in traditional animal models
due to the major differences between human and murine immune systems. The Champions
ImmunoGraft platform has the unique ability of mimicking a human adaptive immune response in
the mice, which allows us to specifically evaluate a variety of cancer therapeutics that modulate
human immunity.

Therapeutic vaccines that trigger the immune system to mount a response against a growing tumor
are another area of intense interest. The development of an effective vaccine remains challenging but
has an outstanding curative potential. Tumors harbor mutations in DNA that result in the translation of
aberrant proteins. While these proteins have the potential to provoke an immune response that
destructs early-stage cancer development, often the immune response becomes insufficient. Vaccines
can trigger it by proactively challenging the system with these specific mutated peptides.
Nevertheless, developing anti-cancer vaccines that effectively inhibit tumor growth has been
complicated, partially due to challenges in finding the critical mutations, among others difficulties.
With the more recent advances in genome sequencing, its now possible to identify tumor-specific
antigens, or neoantigens, that naturally develop as an individuals tumor grows and mutates, she
continued.
Traumatic spinal cord injury in mice with human immune systems.
Carpenter RS, Kigerl KA, Marbourg JM, Gaudet AD, Huey D, Niewiesk S, Popovich PG.
Exp Neurol. 2015 Jul 17;271:432-444. doi: 10.1016/j.expneurol.2015.07.011. [Epub ahead of print]
Inflamm Bowel Dis. 2015 Jul;21(7):1652-73. doi: 10.1097/MIB.0000000000000446.

Use of Humanized Mice to Study the Pathogenesis of Autoimmune and

Inflammatory Diseases.
Koboziev I1, Jones-Hall Y, Valentine JF, Webb CR, Furr KL, Grisham MB.

Author information
Abstract
Animal models of disease have been used extensively by the research community for the past several
decades to better understand the pathogenesis of different diseases and assess the efficacy and
toxicity of different therapeutic agents. Retrospective analyses of numerous preclinical intervention
studies using mouse models of acute and chronic inflammatory diseases reveal a generalized failure to
translate promising interventions or therapeutics into clinically effective treatments in patients.
Although several possible reasons have been suggested to account for this generalized failure to
translate therapeutic efficacy from the laboratory bench to the patients bedside, it is becoming
increasingly apparent that the mouse immune system is substantially different from the human.
Indeed, it is well known that >80 major differences exist between mouse and human immunology; all
of which contribute to significant differences in immune system development, activation, and
responses to challenges in innate and adaptive immunity. This inconvenient reality has prompted

investigators to attempt to humanize the mouse immune system to address important human-specific
questions that are impossible to study in patients. The successful long-term engraftment of human
hematolymphoid cells in mice would provide investigators with a relatively inexpensive small animal
model to study clinically relevant mechanisms and facilitate the evaluation of human-specific therapies
in vivo. The discovery that targeted mutation of the IL-2 receptor common gamma chain in
lymphopenic mice allows for the long-term engraftment of functional human immune cells has
advanced greatly our ability to humanize the mouse immune system. The objective of this review is to
present a brief overview of the recent advances that have been made in the development and use of
humanized mice with special emphasis on autoimmune and chronic inflammatory diseases. In
addition, we discuss the use of these unique mouse models to define the human-specific
immunopathological mechanisms responsible for the induction and perpetuation of chronic gut
inflammation.
J Immunother Cancer. 2015 Apr 21;3:12. doi: 10.1186/s40425-015-0056-2. eCollection 2015.

Human tumor infiltrating lymphocytes cooperatively regulate prostate

tumor growth in a humanized mouse model.
Roth MD1, Harui A1.

Author information
Abstract
BACKGROUND:
The complex interactions that occur between human tumors, tumor infiltrating lymphocytes (TIL) and
the systemic immune system are likely to define critical factors in the host response to cancer. While
conventional animal models have identified an array of potential anti-tumor therapies, mouse models
often fail to translate into effective human treatments. Our goal is to establish a humanized tumor
model as a more effective pre-clinical platform for understanding and manipulating TIL.

METHODS:
The immune system in NOD/SCID/IL-2Rnull (NSG) mice was reconstituted by the co-administration
of human peripheral blood lymphocytes (PBL) or subsets (CD4+ or CD8+) and autologous human
dendritic cells (DC), and animals simultaneously challenged by implanting human prostate cancer cells
(PC3 line). Tumor growth was evaluated over time and the phenotype of recovered splenocytes and
TIL characterized by flow cytometry and immunohistochemistry (IHC). Serum levels of circulating
cytokines and chemokines were also assessed.

RESULTS:
A tumor-bearing huPBL-NSG model was established in which human leukocytes reconstituted
secondary lymphoid organs and promoted the accumulation of TIL. These TIL exhibited a unique
phenotype when compared to splenocytes with a predominance of CD8+ T cells that exhibited
increased expression of CD69, CD56, and an effector memory phenotype. TIL from huPBL-NSG
animals closely matched the features of TIL recovered from primary human prostate cancers. Human
cytokines were readily detectible in the serum and exhibited a different profile in animals implanted
with PBL alone, tumor alone, and those reconstituted with both. Immune reconstitution slowed but
could not eliminate tumor growth and this effect required the presence of CD4+ T cell help.

CONCLUSIONS:
Simultaneous implantation of human PBL, DC and tumor results in a huPBL-NSG model that
recapitulates the development of human TIL and allows an assessment of tumor and immune system
interaction that cannot be carried out in humans. Furthermore, the capacity to manipulate individual
features and cell populations provides an opportunity for hypothesis testing and outcome monitoring
in a humanized system that may be more relevant than conventional mouse models.
Methods Mol Biol. 2014;1213:379-88. doi: 10.1007/978-1-4939-1453-1_31.

A chimeric mouse model to study immunopathogenesis of HCV infection.

Bility MT1, Curtis A, Su L.

Author information
Abstract
Several human hepatotropic pathogens including chronic hepatitis C virus (HCV) have narrow species
restriction, thus hindering research and therapeutics development against these pathogens.
Developing a rodent model that accurately recapitulates hepatotropic pathogens infection, human
immune response, chronic hepatitis, and associated immunopathogenesis is essential for research and
therapeutics development. Here, we describe the recently developed AFC8 humanized liver- and
immune system-mouse model for studying chronic hepatitis C virus and associated human immune
response, chronic hepatitis, and liver fibrosis.
PMID:
25173399
[PubMed indexed for MEDLINE]

PMCID:
PMC4329723
Free PMC Article

Images from this publication.See all images (3)Free text

Immune humanization of immunodeficient mice using diagnostic bone marrow aspirates from
carcinoma patients.
Werner-Klein M, Proske J, Werno C, Schneider K, Hofmann HS, Rack B, Buchholz S, Ganzer R, Blana A,
Seelbach-Gbel B, Nitsche U, Mnnel DN, Klein CA.
PLoS One. 2014 May 15;9(5):e97860. doi: 10.1371/journal.pone.0097860. eCollection 2014.
From 2015 AACR National Meeting in Philadelphia

LB-050: Patient-derived tumor xenografts in humanized NSG mice: a model to study immune
responses in cancer therapy
Sunday, Apr 19, 2015, 3:20 PM 3:35 PM
Minan Wang1, James G. Keck1, Mingshan Cheng1, Danying Cai1, Leonard Shultz2, Karolina
Palucka2, Jacques Banchereau2, Carol Bult2, Rick Huntress2. 1The Jackson Laboratory,
Sacramento, CA; 2The Jackson Laboratory, Bar Harbor, ME

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Other posts on this site on Animal Models, Disease and Cancer Include:

Heroes in Medical Research: Developing Models for Cancer Research

Guidelines for the welfare and use of animals in cancer research
Model mimicking clinical profile of patients with ovarian cancer @ Yale
School of Medicine
Vaccines, Small Peptides, aptamers and Immunotherapy [9]
Immunotherapy in Cancer: A Series of Twelve Articles in the Frontier of
Oncology by Larry H Bernstein, MD, FCAP
Mouse With Humanized Version Of Human Language Gene Provides
Clues To Language Development
The SCID Pig: How Pigs are becoming a Great Alternate Model for
Cancer Research
The SCID Pig II: Researchers Develop Another SCID Pig, And Another
Great Model For Cancer Research

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Steroids, Inflammation, and CAR-T Therapy

September 14, 2015 by sjwilliamspa

Steroids, Inflammation, and CAR-T Therapy [6.3.8]

Reporter: Stephen J. Williams, Ph.D.

Corticosteroids have been used as anticancer agents since the 1940s, with activity reported in a wide
variety of solid tumors, including breast and prostate cancer, and the lymphoid hematologic
malignancies. They are commonly found in regimens for acute lymphocytic leukemia, Hodgkins and
non-Hodgkins lymphoma, myeloma, and chronic lymphocytic leukemia.

A great review on the mechanism of action of prednisones antitumoral activity is seen in

Corticosteroids in the Treatment of Neoplasms Lorraine I. McKay, PhD and John A. Cidlowski,
PhD. in Holland-Frei Cancer Medicine. 6th edition.

It was first discovered that cortisone caused tumor regression in a transplantable mouse
lymphosarcoma,81 a finding soon extended to a wide variety of murine lymphatic tumors. The effects
of corticosteroids were also evaluated on many nonendocrine and nonlymphoid transplantable rodent
tumors. Pharmacologic doses of steroid inhibited growth of various tumor systems. 82 Tissue culture
studies confirmed that lymphoid cells were the most sensitive to glucocorticoids, and responded to
treatment with decreases in DNA, ribonucleic acid (RNA), and protein synthesis. 83 Studies of
proliferating human leukemic lymphoblasts supported the hypothesis that glucocorticoids have
preferential lymphocytolytic effects. The mechanism of action was initially thought to be caused by
impaired energy use via decreased glucose transport and/or phosphorylation; it was later discovered
that glucocorticoids induce apoptosis, or programmed cell death, in certain lymphoid cell
populations.84,85

For review on corticosteroids in cancer therapy see more at:http://www.cancernetwork.com/reviewarticle/corticosteroids-advanced-cancer#sthash.IwHbekuI.dpuf

However, as Dana Farbers Dr. George Canellos, M.D. ponders in Can MOPP be replaced in the
treatment of advanced Hodgkins disease? Semin Oncol. Canellos GP1. 1990 Feb;17(1 Suppl 2):2-6.,
many dose-limiting toxicities occur with MOPP (mechlorethamine, vincristine, procarbazine,
prednisone) therapy used in advanced Hodgkins disease. Although, at the time, he generally was
looking to establish combination therapies with less side effect, the advent of more personalized
therapies as well as immunotherapies may indeed replace the older regimens for B-cell malignancies
and Hodgkins disease, and their panels of toxicities.
Short-term side effects of prednisone (Cancer.gov prednisone description with side effects) as
with all glucocorticoids, include high blood glucose levels (especially in patients with diabetes
mellitus or on other medications that increase blood glucose, such as tacrolimus)

and mineralocorticoid effects such as fluid retention.[10] The mineralocorticoid effects of prednisone are
minor, which is why it is not used in the management of adrenal insufficiency, unless a more potent
mineralocorticoid is administered concomitantly.
Long-term side effects include Cushings syndrome, steroid dementia syndrome, truncal weight
gain, osteoporosis, glaucoma and cataracts, type II diabetes mellitus, and depression upon dose
reduction or cessation.
Therefore the oncology world has been moving toward therapies which are more selective with less
dose-limiting toxicities (e.g. Rituximab), and are looking to CAR-T therapies as a possible replacement
for standard chemotherapeutic regimens. However, as with prednisone, there have been serious
adverse events in some CAR-T clinical trials. Luckily clinicians, as discussed below, have found
supportive therapies to alleviate the most severe side effects to CAR-T.
This section will be refer to supportive therapies as those adjuvant therapy given to
alleviate patient discomfort, reduce toxicities and adverse event, or support patient wellbeing during their course of chemotherapy, not adjuvant therapy to enhance antitumoral
effect.
For more background information of CAR-T therapies and related issues please see my
previous post
NIH Considers Guidelines for CAR-T therapy: Report from Recombinant DNA Advisory Committee
The following is a brief re-post of some of the important points for reference to this new posting.

1. Evolution of Chimeric Antigen Receptors

Early evidence had suggested that adoptive transfer of tumor-infiltrating lymphocytes, after depletion
of circulating lymphocytes, could result in a clinical response in some tumor patients however
developments showed autologous T-cells (obtained from same patient) could be engineered to express
tumor-associated antigens (TAA) and replace the TILS in the clinical setting.

A brief history of construction of 2nd and 3rd generation CAR-T cells given by cancer.gov:
http://www.cancer.gov/cancertopics/research-updates/2013/CAR-T-Cells

Differences between second- and third-generation chimeric antigen receptor T

cells. (Adapted by permission from the American Association for Cancer Research: Lee, DW et al. The
Future Is Now: Chimeric Antigen Receptors as New Targeted Therapies for Childhood Cancer. Clin
Cancer Res; 2012;18(10); 278090. doi:10.1158/1078-0432.CCR-11-1920)

Constructing a CAR T Cell (from cancer.gov)

The first efforts to engineer T cells to be used as a cancer treatment began in the early 1990s. Since
then, researchers have learned how to produce T cells that express chimeric antigen receptors (CARs)
that recognize specific targets on cancer cells.
The T cells are genetically modified to produce these receptors. To do this, researchers use viral
vectors that are stripped of their ability to cause illness but that retain the capacity to integrate into
cells DNA to deliver the genetic material needed to produce the T-cell receptors.
The second- and third-generation CARs typically consist of a piece of monoclonal antibody, called a
single-chain variable fragment (scFv), that resides on the outside of the T-cell membrane and is linked
to stimulatory molecules (Co-stim 1 and Co-stim 2) inside the T cell. The scFv portion guides the cell
to its target antigen. Once the T cell binds to its target antigen, the stimulatory molecules provide the
necessary signals for the T cell to become fully active. In this fully active state, the T cells can more
effectively proliferate and attack cancer cells.

2. Consideration for Design of Trials and Mitigating Toxicities

Early Toxic effects Cytokine Release Syndrome The effectiveness of CART

therapy has been manifested by release of high levels of cytokines resulting in fever
and inflammatory sequelae. One such cytokine, interleukin 6, has been attributed to
this side effect and investigators have successfully used an IL6 receptor
antagonist, tocilizumab (Acterma), to alleviate symptoms of cytokine release
syndrome (see review Adoptive T-cell therapy: adverse events and safety switches by
Siok-Keen Tey).

Early Toxic effects Over-activation of CAR T-cells; mitigation by dose escalation

strategy (as authors in reference [3] proposed). Most trials give billions of genetically
modified cells to a patient.

Late Toxic Effects long-term depletion of B-cells . For example CART directing
against CD19 or CD20 on B cells may deplete the normal population of CD19 or CD20
B-cells over time; possibly managed by IgG supplementation

References
1.

Ertl HC, Zaia J, Rosenberg SA, June CH, Dotti G, Kahn J, Cooper LJ, Corrigan-Curay
J, Strome SE: Considerations for the clinical application of chimeric antigen
receptor T cells: observations from a recombinant DNA Advisory Committee
Symposium held June 15, 2010. Cancer research 2011,71(9):3175-3181.

2.

Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA: Case
report of a serious adverse event following the administration of T cells
transduced with a chimeric antigen receptor recognizing ERBB2. Molecular
therapy : the journal of the American Society of Gene Therapy 2010, 18(4):843851.

3.

Kandalaft LE, Powell DJ, Jr., Coukos G: A phase I clinical trial of adoptive
transfer of folate receptor-alpha redirected autologous T cells for recurrent
ovarian cancer. Journal of translational medicine 2012, 10:157.

3. Case Reports of Adverse Events and Their Amelioration During CAR-T

Therapy
CAR-T Therapy have Had reports of Serious Adverse Events

From FierceBiotech UPDATED: Two

deaths force MSK to hit the brakes on
engineered T cell cancer study
April 6, 2014 | By John Carroll
Safety concerns forced investigators at Memorial Sloan-Kettering Cancer Center to suspend patient
recruitment for an early-stage study of a closely watched approach to reengineering the immune
system to fight cancer. Several days ago MSK updated a site on clinicaltrials.gov to note that it was

halting recruitment for a small study using T cells reengineered with chimeric antigen receptors
(CARs) against CD19-positive B cells for aggressive non-Hodgkin lymphoma, triggering concerns about
the potential fallout at Juno Therapeutics, the biotech formed to commercialize the effort. And Sunday
evening representatives for MSK revealed at the meeting of the American Association for Cancer
Research in San Diego that the deaths of two patients spurred investigators to rethink the trial
protocol on recruitment, revamping the patient profile to account for the threat of comorbidities while
adjusting the dose based on the extent of disease at the time of treatment.
For more on this story please see
Source: http://www.fiercebiotech.com/story/memorial-sloan-kettering-hits-brakes-engineered-t-cellcancer-study/2014-04-06

Keynote presentation by Carl H. June, recipient of The Richard V. Smalley MD 2013 Award

As reported in 2013 in Highlights and summary of the 28th annual meeting of the Society for
Immunotherapy of Cancer by Paolo A Ascierto1, David H Munn2,Anna K Palucka34 and Paul M
Sondel in Journal of ImmunoTherapy of Cancer

Since 2005, SITC honors a luminary in the field who has significantly contributed to the advancement
of cancer immunotherapy research by presenting the annual Richard V. Smalley MD Memorial Award,
which is associated with the Smalley keynote lecture at the Annual SITC meeting. The awardee this
year Carl H. June of the University of Pennsylvania, has led innovative translational research for over
25 years, with the most recent focus being the development of the Chimeric Antigen Receptor
modified T-cell (CART) approach. Carl June summarized how the past 15 years of progress have
expanded upon the original concept presented by Zelig Eshhar [4], in which variable regions of tumorreactive monoclonal antibodies (mAbs) (VH and VL) are linked to transmembrane and signaling
domains of T cell activating molecules to create membrane based receptors with specificity for the
tumor antigen recognized by the original mAb [4]. These receptors can be transfected into T cells, for
example with lentiviruses. Pre-clinical work demonstrated how CD3- and 41BB signaling components
enhanced proliferation and survival of T cells in hypoxic conditions. The initial clinical work has been
done with CART reactive to CD-19 on malignant B cells, with progress particularly in chronic
lymphocytic leukemia (CLL) in adults and acute lymphoblastic leukemia (ALL) in children [5,6]. As of

the SITC meeting, CarlJunes team had treated 35 patients with CLL and 20 with ALL. Of the 20 with
ALL, had relapsed after allogeneic BMT. Of these 20 children, 17 were in complete remission, and
with persistent B cell aplasia; documenting the persistent effects of the CART cells. Toxicities included
the persistent B cell aplasia and profound tumor lysis and cytokine storm, seen 12 weeks into the
treatment for ALL. This cytokine storm has been ameliorated by using anti-IL6 mAb. The B cell
aplasia, while undesired, is acceptable, as patients can receive passive replacement of IgG, thus
making their B cells expendable. These CART cells can traffic into the CNS. In ALL patients, it
appears that each individual CART cell (or its progeny) can destroy 1000 tumor cells. Ongoing efforts
in CarlJunes program, and at other centers, are now moving into analyses of CART reactive with other
tumor targets, by using mAbs that recognize antigens expressed on other tumors. Among these are
EGFR on glioblastoma, PSMA on prostate cancer, mesothelin on ovarian cancer, HER2 on breast (and
other) cancers, and several other targets. Because some of these targets are also expressed on
normal tissues that are not expendable, novel approaches are being developed to decrease the
potency or longevity of the CART effect, to decrease potential toxicity. This includes generating short
lived CART cells by inducing CAR expression with short-lived RNA, rather than transfecting with a DNA
construct that remains permanently.

In T-Cell Immunotherapy: Looking Forward Molecular Therapy (2014); 22 9, 15641574.

doi:10.1038/mt.2014.148 many of the leading CAR-T clinicians and investigators reported on some of
the adverse events related o CAR-T therapy including

40 severe adverse events (SAE) had been reported from 2010 to 2013.

B-cell aplasia

Systemic inflammatory release syndrome (CRS) {the most sever toxicity seen}

Tumor lysis syndrome

CNS toxicity

Macrophage activation syndrome

According to the investigators the systemic inflammatory release syndrome (CRS) is the most severe
toxicity seen

The most commonly reported adverse event is CRS,49 with about three-quarters of the patients with
CRS requiring admission to an intensive care unit. In the case of CAR therapy, the onset of CRS is
related to the particular signaling domain in the CAR, with early-onset CRS in the first several days

after infusion related to CARs that encode a CD28 signaling domain. 4,16 By contrast, CARs encoding a
4-1BB signaling domain tend to have delayed-onset CRS (range, 7 to 50 days) after CAR T-cell
infusion.6 CRS has also been reported after the infusion of TCR-modified T cells, with onset typically
five to seven days after infusion. The development of CRS is often, but not invariably, associated with
clinically beneficial tumor regression. Several cytokines have been reported to be elevated in the
serummost commonly, interferon (IFN)-, tumor necrosis factor (TNF)-, and interleukin (IL)6. Management of CRS has included supportive care, corticosteroids, etanercept,
tocilizumab, and alemtuzumab. The role of suicide genes in the management of CRS remains
unknown.50

This supportive therapy have now been included in all protocols now and sites are engaged in
developing pharmacovigilance protocol development for CAR-T therapy.

Other posts on this site on Immunotherapy and Cancer include

Combined anti-CTLA4 and anti-PD1 immunotherapy shows promising
results against advanced melanoma
Molecular Profiling in Cancer Immunotherapy: Debraj GuhaThakurta, PhD
Pancreatic Cancer: Genetics, Genomics and Immunotherapy
$20 million Novartis deal with University of Pennsylvania to develop
Ultra-Personalized Cancer Immunotherapy
Upcoming Meetings on Cancer Immunogenetics
Tang Prize for 2014: Immunity and Cancer
ipilimumab, a Drug that blocks CTLA-4 Freeing T cells to Attack Tumors
@DM Anderson Cancer Center
Junos approach eradicated cancer cells in 10 of 12 leukemia patients,
indicating potential to transform the standard of care in oncology
Report on Cancer Immunotherapy Market & Clinical Pipeline Insight
New Immunotherapy Could Fight a Range of Cancers

Posted in Cancer and Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy,FDA, FDA Regulatory
Affairs, Immuno-Oncology & Genomics, Innovation in immunology diagnostics, lymphoma, Personal Health
Applications: Tech Innovations serves HealhCare,Personalized and Precision Medicine & Genomic Research |
Tagged acute and chronic leukemias, b cell lymphoma, cancer, Cancer immunotherapy, CAR-T, chemotherapeutic

toxicity, Conditions and Diseases, FDA, Food and Drug Administration, Hodgkin's lymphoma,medicine, Non-Hodgkin
lymphoma, Personalized medicine, research, toxicity | Leave a comment

Envisage-Wistar Partnership and Immunacel LLC Presents at PCCI

June 3, 2015 by sjwilliamspa

The Pharmaceutical Consulting Consortium International (PCCI) June Meeting:

Envisage-Wistar Partnership and Immunacel LLC
An early stage healthcare venture creation and management firm
Presenter: Vic Subbu, COO of Immunacel & Managing Partner of Envisage and Heather Steinman, VP
of Business Development & Executive Director Tech Transfer Wistar Institute
Monday, June 8, 2015
Embassy Suites, Chesterbrook, Pennsylvania (directions)
Announcement from the PCCI website:
Much has been said lately about how to improve the tech transfer situation. Wistar is
meeting this challenge. Immunacel is the first of a series of developmental challenges and
the Envisage-Wistar partnership solution becomes the meat of the evenings discussion.
The Wistar Institute is the nations first independent institution devoted to medical research and
training. The Wistar Institute has evolved from its beginnings as an anatomical teaching museum to its
present-day status as an international leader in basic biomedical research.
Envisage LLC is an early stage healthcare venture creation and management firm. By focusing on
key healthcare segments, Envisage aims to identify and advance promising healthcare innovations
into value-add ventures.
IMMUNACCEL LLC is a Wistar Institute spin-out focused on accelerating the development of
immune-mediated treatments for cancer and other unmet medical needs:
MMUNACCELs 3-D cancer-immune cell organotypic culture system is a physiologically relevant culture
system utilizing primary human cancer cells and cytotoxic T cells (CTL) generated from patient T-cells,
amongst fibroblasts and collagen assembled in a 3-D organotypic model.
Other related articles on PCCI and Philadelphia Biotech were published in this Open Access
Online Scientific Journal, include the following:

PCCIs 7th Annual Roundtable Crowdfunding for Life Sciences: A Bridge Over Troubled
Waters? May 12 2014 Embassy Suites Hotel, Chesterbrook PA 6:00-9:30 PM

Protecting Your Biotech IP and Market Strategy: Notes from Life Sciences Collaborative
2015 Meeting
The Vibrant Philly Biotech Scene: Focus on KannaLife Sciences and the Discipline and
Potential of Pharmacognosy
The Vibrant Philly Biotech Scene: Focus on Computer-Aided Drug Design and Gfree Bio, LLC
The Vibrant Philly Biotech Scene: Focus on Vaccines and Philimmune, LLC
The Bioscience Crowdfunding Environment: The Bigger Better VC?
R&D Alliances between Big Pharma and Academic Research Centers: Pharmas Realization
that Internal R&D Groups alone arent enough
BIO Partnering: Intersection of Academic and Industry: BIO INTERNATIONAL CONVENTION
June 23-26, 2014 | San Diego, CA
Diagnostics and Biomarkers: Novel Genomics Industry Trends vs Present Market Conditions
and Historical Scientific Leaders Memoirs
Posted in Global Partnering & Biotech Investment, Immuno-Oncology & Genomics,Innovation in immunology
diagnostics, Intellectual Property, Innovations, Commercialization, Investment in technological
breakthrough, Investment in Technological Breakthrough, Medical Devices R&D Investment, Organ-on-a-Chip & 3D
Printing in Life Sciences, Personalized and Precision Medicine & Genomic Research, Pharmaceutical R&D
Investment, Pharmaceuticall R&D Informatics, Scientific & Biotech Conferences: Press Coverage, Small Molecules in
Development of Therapeutic Drugs | Tagged 3D-biomatrix,bioinvestment, cancer, Cancer immunotherapy, CART, Clinical trial, crowd-funding,organotypical culture, Personalized medicine, Publicprivate
partnership, research | Leave a comment

Vaccines, Small Peptides, aptamers and Immunotherapy [9]

May 12, 2015 by larryhbern

Vaccines, Small Peptides, aptamers and Immunotherapy [9]

Writer and Curator: Larry H. Bernstein, MD, FCAP
This contribution has the following structure:
9.1.1 Viruses in carcinogenesis
9.1.2

Simultaneous Humoral and Cellular Immune Response against CancerTestis Antigen NY-ESO-

1: Definition of Human Histocompatibility Leukocyte Antigen (HLA)-A2binding Peptide Epitopes

9.1.3 Monoclonal Antibodies in Cancer Therapy
9.1.4 Aptamers
9.1.5 Tumor Suppressors

9.1 Vaccines
9.1.1 Viruses in carcinogenesis

HPV-associated cervical cancer

HPV-associated head and neck cancer: a virus-related cancer epidemic

The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver
cancer worldwide
Risk of pancreatic cancer among individuals with hepatitis C or hepatitis B virus infection: a nationwide
study in Sweden.
HIV Infection and Cancer Risk
HIV and cancer of the cervix
Anal cancer: an HIV-associated cancer
The therapeutic potential of CXCR4 antagonists in the treatment of HIV infection, cancer metastasis
and rheumatoid arthritis
Types of Cancer: AIDS/HIV related malignancies
Cytokines in cancer pathogenesis and cancer therapy
Dendritic Cells as Therapeutic Vaccines against Cancer
9.1.2

Simultaneous Humoral and Cellular Immune Response against CancerTestis Antigen

NY-ESO-1: Definition of Human Histocompatibility Leukocyte Antigen (HLA)-A2binding

Peptide Epitopes
9.1.3 Monoclonal antibodies
Monoclonal antibodies in cancer therapy
Monoclonal Antibodies in Cancer Therapy: 25 Years of Progress
9.1.4 Aptamers
Nanocarriers as an emerging platform for cancer therapy
Quantum DotAptamer Conjugates for Synchronous Cancer Imaging, Therapy, and Sensing of Drug
Delivery Based on Bi-Fluorescence Resonance Energy Transfer
Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

9.1 Vaccines
9.1.1 Viruses in carcinogenesis
HPV-associated cervical cancer
http://www.cancer.net/navigating-cancer-care/prevention-and-healthy-living/hpv-and-cancer
Human papillomavirus (HPV) is a virus that is usually passed on during direct skin-to-skin contact,
most commonly sex. In fact, HPV is the most common sexually transmitted disease in the United
States. Most men and women are not aware they have an HPV infection because they do not develop
any symptoms or health problems. Certain HPV types can cause precancerous lesions (areas of
abnormal tissue) or cancer.
More than 40 of the viruses are called genital type HPVs. These viruses are spread from person to
person when their genitals come into contact, usually during vaginal or anal sex. They can also be
passed on through oral sex.
Genital HPV types can infect the genital area of women, including the vulva (outer portion of the
vagina), the vagina, and the cervix (the lower, narrow part of a womans uterus), as well as the genital
area of men, including the penis. In both men and women, genital HPV can infect the anus and some
areas of the head and neck.
Nearly all cervical cancers are caused by HPV infection. Strong scientific evidence shows that a lasting
HPV infection is required for cervical cancer to begin developing. Whether a woman who is infected
with HPV will develop cervical cancer depends on a number of factors, including the type of HPV
infection she has. Of the cervical cancers related to HPV, about 70% are caused by two strains, HPV16 or HPV-18. In women who have HPV, smoking may increase the risk of cervical cancer.
Warts and precancerous lesions can be removed through cryotherapy (freezing); loop electrosurgical
excision procedure (LEEP), which uses electric current to remove abnormal tissue; or surgery.
Receiving an HPV vaccine reduces your risk of infection. The U.S. Food and Drug Administration (FDA)
approved two vaccines that help prevent HPV infection: Gardasil and Cervarix. It is important to note
that the vaccines cannot cure an existing HPV infection.
Purpose of the vaccines. The goal of HPV vaccination is to prevent a lasting HPV infection after a
person is exposed to the virus. Gardasil, introduced in 2006, helps prevent infection from the two
HPVs known to cause most cervical cancers and precancerous lesions in the cervix. The vaccine also
prevents against the two low-risk HPVs known to cause 90% of genital warts. Gardasil is approved for

the prevention of cervical, vaginal, and vulvar cancers in girls and women ages nine to 26. It is also
approved to prevent anal cancer in women and men and genital warts in men and boys in the same
age range. Cervarix, introduced in 2009, is approved for the prevention of cervical cancer in girls and
women ages 10 to 25.
Effectiveness and safety of the vaccines. Data show the HPV vaccinations are safe and highly
effective in preventing a lasting infection of the HPV types they target. Because it takes many years
before a precancerous lesion develops into an invasive cancer, it will likely take several more years
before there is evidence that the number of cancer cases in vaccinated individuals has been reduced.
HPV-associated head and neck cancer: a virus-related cancer epidemic
Shanthi Marur, Gypsyamber DSouza, William H Westra, Arlene A Forastiere
Lancet Oncol 2010; 11: 78189
http://dx.doi.org:/10.1016/S1470-2045(10)70017-6
A rise in incidence of oropharyngeal squamous cell cancerspecifically of the lingual and palatine
tonsilsin white men younger than age 50 years who have no history of alcohol or tobacco use has
been recorded over the past decade. This malignant disease is associated with human papillomavirus
(HPV) 16 infection. The biology of HPV-positive oropharyngeal cancer is distinct with P53 degradation,
retinoblastoma RB pathway inactivation, and P16 upregulation. By contrast, tobacco-related
oropharyngeal cancer is characterized by TP53 mutation and downregulation of CDKN2A (encoding
P16). The best method to detect virus in tumor is controversial, and both in-situ hybridization and PCR
are commonly used; P16 immunohistochemistry could serve as a potential surrogate marker. HPVpositive oropharyngeal cancer seems to be more responsive to chemotherapy and radiation than HPVnegative disease. HPV 16 is a prognostic marker for enhanced overall and disease-free survival, but its
use as a predictive marker has not yet been proven. Many questions about the natural history of oral
HPV infection remain under investigation. For example, why does the increase in HPV-related
oropharyngeal cancer dominate in men? What is the potential of HPV vaccines for primary prevention?
Could an accurate method to detect HPV in tumor be developed? Which treatment strategies reduce
toxic effects without compromising survival? Our aim with this review is to highlight current
understanding of the epidemiology, biology, detection, and management of HPV-related oropharyngeal
head and neck squamous cell carcinoma, and to describe unresolved issues.
Cancers of the head and neck arise from mucosa lining the oral cavity, oropharynx, hypopharynx,
larynx, sinonasal tract, and nasophaynx. By far the most common histological type is squamous cell
carcinoma, and grade can vary from well-differentiated keratinizing to undifferentiated non-

keratinizing. An increase in incidence of oropharyngeal squamous cell carcinomaspecifically in the

tonsil and tongue basehas been seen in the USA, most notably in individuals aged 4055 years.
Patients with oropharyngeal cancer are mainly white men. Unlike most tobacco-related head and neck
tumors, patients with oropharyngeal carcinoma usually do not have a history of tobacco or alcohol
use. Instead, their tumors are positive for oncogenic forms of the human papillomavirus (HPV),
particularly 16 type. About 60% of oropharyngeal squamous cell cancers in the USA are positive for
HPV 16. HPV-associated head and neck squamous cell carcinoma seems to be a distinct clinical entity,
and this malignant disease has a better prognosis than HPV-negative tumors, due in part to increased
sensitivity of cancers to chemotherapy and radiation therapy. Although HPV is now recognized as a
causative agent for a subset of oropharyngeal squamous cell carcinomas, the biology and natural
history of oropharyngeal HPV infection and the best clinical management of patients with HPV-related
head and neck squamous cell tumors is not well understood.
Head and neck cancer is the sixth most common cancer worldwide, with an estimated annual burden
of 563 826 incident cases (including 274 850 oral cavity cancers, 159 363 laryngeal cancers, and 52
100 oropharyngeal cancers) and 301 408 deaths. 1 Although HPV has been long known to be an
important cause of anogenital cancer, only in recent times has it been recognized as a cause of a
subset of head and neck squamous cell carcinomas. 2 More than 100 different types of HPV exist,3 and
at least 15 types are thought to have oncogenic potential.4 However, most (>90%) HPV-associated
head and neck squamous cell cancers are caused by one virus type, HPV 16, the same type that leads
to HPV-associated anogenital cancers. The proportion of head and neck squamous cell carcinomas
caused by HPV varies widely (figure 1),516 largely because of the burden of tobacco-associated disease
in this population of tumors. Tobacco, alcohol, poor oral hygiene, and genetics remain important risk
factors for head and neck tumors overall, but HPV is now recognized as one of the primary causes of
oropharyngeal squamous cell cancers. In the USA, about 4080% of oropharyngeal cancers are
caused by HPV, whereas in Europe the proportion varies from around 90% in Sweden to less than 20%
in communities with the highest rates of tobacco use (figure 1).
The incidence of head and neck cancers overall in the USA has fallen in recent years, consistent with
the decrease in tobacco use in this region. By contrast, incidence of HPV-associated oropharyngeal
cancer seems to be rising, highlighting the increasing importance of this causal association. 1719 In a
US study in which data of the Surveillance, Epidemiology, and End Results (SEER) program were used,
incidence of oropharyngeal tumors (which are most likely to be HPV-associated) rose by 13% for base
of tongue cancers and by 06% for tonsillar cancers every year between 1973 and 2004. By contrast,
incidence of oral cavity cancers (not associated with HPV) declined by 19% every year during the

same period.17 The age-adjusted incidence of tonsillar cancer increased 35-fold in women and 26-fold
in men between 1970 and 2002.24 Augmented incidence of HPV-associated oropharyngeal cancers
represents an emerging viral epidemic of cancer.
Why is increased incidence of HPV-associated oropharyngeal cancer most pronounced in young
individuals? This effect could be attributable to changes in sexual norms (i.e., more oral sex partners
or oral sex at an earlier age in recent than past generations) combined with fewer tobacco-associated
cancers in young cohorts, making the outcomes of HPV-positive cancers more visible. Can the higher
rates of HPV-associated oropharyngeal cancers in men compared with women be accounted for solely
by differences in sexual behavior, or are biological differences in viral clearance present that could
contribute to the higher burden of these cancers in men? HPV prevalence in cervical rather than penile
tissue might boost the chances of HPV infection when performing oral sex on a woman, contributing to
the higher rate of HPV-associated oropharyngeal cancer in men.
Tobacco use has fallen in past decades, and the corresponding rise in proportion of head and neck
cancers that are oropharyngeal in origin has been striking, both in the USA and internationally. SEER
data suggest that about 18% of all head and neck carcinomas in the USA were located in the
oropharynx in 1973, compared with 31% of such squamous cell tumors in 2004. 19 Similarly, in
Sweden, the proportion of oropharyngeal cancers caused by HPV has steadily increased, from 23% in
the 1970s to 57% in the 1990s, and as high as 93% in 2007.13,25 These data indicate that HPV is
now the primary cause of tonsillar malignant disease in North America and Europe.
Findings of initial studies suggest that oral HPV frequency increases with age. Prevalent oral HPV
infection is detected in 35% of adolescents 2628 and 510% of adults.14,29 We do not yet know whether
the natural history of oral HPV or risk factors for persistent HPV infection in the oropharynx differ from
those known for anogenital HPV infection (table 1). Data suggest oral HPV prevalence is amplified with
number of sexual partners and is more typical in men, in HIV-infected individuals, and in current
tobacco users.2628,30,31
In view of the importance of tobacco use in head and neck squamous cell carcinoma, most cases of
this malignant disease seen in non-smokers are unsurprisingly HPV-related. However, oral HPV
infection is common in smokers and non-smokers and is an important cause of oropharyngeal cancer
in both groups. For example, in case series, only 1316% of individuals with HPV-positive head and
neck squamous cell cancer did not smoke or drink alcohol. 32,33 Although a higher proportion of
individuals with HPV-positive compared with HPV-negative tumors are non-smokers or neither smoke
nor drink alcohol, many with HPV-positive disease have a history of alcohol and tobacco use. In fact,

1030% of HPV-positive head and neck squamous cell carcinomas were recorded in heavy tobacco
and alcohol users.32,33 This finding underscores that HPV-associated malignant disease not only arises
in people who do not smoke or drink alcohol but also occurs in people with the traditional risk factors
of tobacco and alcohol use.
HPV detection may ultimately serve a more comprehensive role than mere prognostication. Detection
of HPV is emerging as a valid biomarker for discerning the presence and progress of disease
encompassing all aspects of patients care, from early cancer detection,41 to more accurate tumor
staging (e.g., localization of tumor origin),42,43 to selection of patients most likely to benefit from
specific treatments,44 to post-treatment tumor surveillance.45,46 Consequently, there is a pressing need
for a method of HPV detection that is highly accurate, reproducible from one diagnostic laboratory to
the next, and practical for universal application in the clinical arena. Despite growing calls for routine
HPV testing of all oropharyngeal carcinomas, the best method for HPV detection is not established.
Various techniques are currently in use, ranging from consensus and type-specific PCR methods, realtime PCR assays to quantify viral load, type-specific DNA in-situ hybridization, detection of serum
antibodies directed against HPV epitopes, and immunohistochemical detection of surrogate biomarkers
(e.g., P16 protein). Although PCR-based detection of HPV E6 oncogene expression in frozen tissue
samples is generally regarded as the gold standard for establishing the presence of HPV, selection of
assays for clinical use will ultimately be influenced by concerns relating to sensitivity, specificity,
reproducibility, cost, and feasibility. Development of non-fluorescent chromogens has enabled
visualization of DNA hybridization by conventional light microscope; furthermore, adaptation of in-situ
hybridization to formalin-fixed and paraffin-embedded tissues has made this technique compatible
with standard tissue-processing procedures and amenable to retrospective analysis of archival tissue
blocks. Most PCR-based methods, on the other hand, need a high level of technical skill and are best
used with fresh-frozen samples.
Limitations of any one detection assay can be offset by algorithms that combine the strengths of
complementary assays.50 A highly feasible strategy incorporates P16 immunohistochemistry and HPV
in-situ hybridization. In view of sensitivity that approaches 100%, P16 immunostaining is a good firstline assay for elimination of HPV-negative cases from any additional analysis. Since specificity is
almost 100%, a finding positive for HPV 16 on in-situ hybridization reduces the number of falsepositive cases by P16 staining alone. A P16-positive, HPV 16-negative result singles out a subset of
tumors that qualifies for rigorous analysis for other (i.e., non-HPV 16) oncogenic virus types.
HPV in-situ hybridisation and P16 immunostaining as a practical diagnostic approach to discernment of
HPV status can be applied readily to cytological preparations, including fi ne-needle aspirates from

patients with cervical lymph-node metastases.41,52 Further expansion of HPV testing to blood and other
body fl uids would advance the role of HPV as a clinically relevant biomarker, but these specimens
would need other detection platforms. PCR-based detection of HPV DNA in blood (53) and saliva (54)
of patients after treatment of their HPV-positive cancers suggests a future role in tumour surveillance.
Detection of serum antibodies to HPV-related epitopes can predict the HPV status of head and neck
cancers, and this method has been advocated as a way to project clinical outcomes and guide
treatment without the constraints of tissue acquisition. 53,55
The increasing prevalence of oropharyngeal cancer in young populations and substantially amplified
survival rates with current treatment approaches stands in contrast to survival achieved in older
individuals with comorbid disorders associated with tobacco and alcohol history. Several characteristics
of patients with head and neck cancer have been linked with favorable prognosis, including nonsmoker, minimum exposure to alcohol, good performance status, and no comorbid disorders, all of
which are related to HPV-positive tumor status. Findings of retrospective analyses suggest that
individuals with HPV-positive oropharyngeal cancer have higher response rates to chemotherapy and
radiation and increased survival6265 compared with those with HPV-negative tumors. Augmented
sensitivity to chemotherapy and radiotherapy has been attributed to absence of exposure to tobacco
and presence of functional unmutated TP53. 63,64,66 Increased survival of patients with HPV-positive
cancer is also possibly attributable in part to absence of field cancerization related to tobacco and
alcohol exposure.67
Survival outcomes for individuals with HPV 16-positive and P16-positive oropharyngeal tumors were
similar. Failure data indicated significantly diminished rates of locoregional failure and second primary
tumour in patients with HPV-positive oropharyngeal cancer compared with those with HPV-negative
tumors; distant metastases did not differ between the two groups. When survival was assessed after
adjustment for tobacco exposure, in individuals who smoked, those with HPV-positive oropharyngeal
tumors and fewer than 20 pack-years had 2-year overall survival of 95%, compared with 80% in those
with HPV-positive cancers and 20 pack-years or more, and 63% in HPV-negative cancers and 20 packyears or more. By comparison with people with HPV-positive oropharyngeal tumors who smoked and
had fewer than 20 pack-years, the hazard of death was raised for those with HPV-negative tumors and
20 pack-years or more (hazard ratio 433) and those with HPV-positive cancers and 20 pack-years or
more (179). These data indicate clearly that tobacco exposure alters the biology of HPV-positive
oropharyngeal tumors and is an important prognostic factor.

An association between HPV-positive, P16-positive oropharyngeal tumors and survival outcomes was
reported in another retrospective analysis of a large phase 3 trial of chemoradiation, which included
more than 800 patients enrolled from international sites.72 This substudy analysis looked at 195
available tumor samples in patients with an oropharyngeal primary cancer, of which 28% were HPVpositive and 58% were P16-positive. Individuals with HPV-positive cancers had 2-year overall survival
of 94% and 2-year failure-free survival of 86% compared with 77% (p=0007) and 75% (p=0035),
respectively, in those with HPV-negative tumors. When co-expression of HPV and P16 was correlated
with survival outcomes, individuals with HPV-positive, P16-positive tumors had 2-year overall survival
of 95% compared with 88% in those with HPV-negative, P16-positive cancers and 71% (p=0003) in
those with HPV-negative, P16-negative tumors. Similar results were noted for 2-year failure-free
survival (89%, 86%, and 69%, respectively; p=0002) and time to locoregional failure (93%, 95%,
and 84%, respectively; p=0051). By multivariable analysis, HPV 16 and P16 were identified as
independent prognostic factors.
ECOG proposes induction chemotherapy with a triple drug regimen to reduce tumor burden to
subclinical disease (clinical complete response at primary site) followed by lower dose radiation (total
dose 54 Gy) and concurrent cetuximab. Overall survival and progression-free survival outcomes will be
assessed and compared with results of the 2008 ECOG study.70 The main aim of this planned study is
to assess potential for a lower dose of radiation to control disease and to investigate toxic effects and
quality-of-life variables.
In summary, tumor HPV status is a prognostic factor for overall survival and progression-free survival
and might also be a predictive marker of response to treatment. The method of in-situ hybridization
provides a feasible approach for implementation in most diagnostic pathology laboratories, and
immunohistochemical staining for P16 could be useful as a surrogate marker for HPV status.
Seemingly, locoregional recurrencebut not the rate of distant diseaseis diminished in patients with
HPV-positive tumors. Smoking and tobacco exposure might modify survival and recurrence of HPVpositive tumors and should be considered in future trials for risk stratification of patients with HPVpositive malignant disease.
HCV and cancer
The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and
primary liver cancer worldwide
Joseph F. Perz, Armstrong GL, Farrington LA, Hutin YJF, Bell BP

J Hepatol 2006; 45:529-538

http://dx.doi.org:/10.1016/j.jhep.2006.05.013
End-stage liver disease accounts for one in forty deaths worldwide. Chronic infections with hepatitis B
virus (HBV) and hepatitis C virus (HCV) are well-recognized risk factors for cirrhosis and liver cancer,
but estimates of their contributions to worldwide disease burden have been lacking. Methods: The
prevalence of serologic markers of HBV and HCV infections among patients diagnosed with cirrhosis or
hepatocellular carcinoma (HCC) was obtained from representative samples of published reports.
Attributable fractions of cirrhosis and HCC due to these infections were estimated for 11 WHO-based
regions. Results: Globally, 57% of cirrhosis was attributable to either HBV (30%) or HCV (27%) and
78% of HCC was attributable to HBV (53%) or HCV (25%). Regionally, these infections usually
accounted for >50% of HCC and cirrhosis. Applied to 2002 worldwide mortality estimates, these
fractions represent 929,000 deaths due to chronic HBV and HCV infections, including 446,000 cirrhosis
deaths (HBV: n = 235,000; HCV: n = 211,000) and 483,000 liver cancer deaths (HBV: n = 328,000;
HCV: n = 155,000). Conclusions: HBV and HCV infections account for the majority of cirrhosis and
primary liver cancer throughout most of the world, highlighting the need for programs to prevent new
infections and provide medical management and treatment for those already infected.
Among primary liver cancers occurring worldwide, hepatocellular carcinoma (HCC) represents the
major histologic type and likely accounts for 70% to 85% of cases [2]. Cirrhosis precedes most cases
of HCC, and may exert a promotional effect via hepatocyte regeneration [3,4]. Compared with other
causes of cirrhosis, chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) is
associated with a higher risk of developing HCC [3,5]. Alcohol abuse represents a leading cause of
cirrhosis and is also a major contributor. dietary aflatoxin exposure in parts of Africa and Asia has been
associated with primary liver cancer, especially in hosts with chronic HBV infection [8].
An understanding of the relative contribution of various etiologies to disease burden is important for
setting public health priorities and guiding prevention programs [10,11]. The World Health
Organizations Global Burden of Disease (GBD) 2000 project aims to quantify the burden of premature
morbidity and mortality from over 130 major causes [1,12]. Liver cancer and cirrhosis are included in
the analysis, but with the exception of alcohol, the etiologies underlying these diseases have not been
well accounted for [1,11,13]. In particular, HBV and HCV infections have been poorly characterized in
previous WHO estimates since these were based primarily on the acute effects of infection and omitted
the associated burdens of chronic liver disease [10,11].

The attributable fraction represents the proportion of disease occurrence that potentially would be
prevented by eliminating a given risk factor. For cirrhosis, a systematic analysis of attributable
fractions has been lacking altogether. For HCC, previous estimates of the attributable fractions due to
HBV and HCV are available but are not comprehensive and do not correspond to the regional
designations and related conventions of the current GBD project [14].
The prevalence of HBV and HCV infection among cirrhosis and HCC patients varied considerably within
and between regions (Tables 2 and 3). These variations tended to reflect known patterns of HBV and
HCV infection endemicity [99,100]. For example, in countries where HCV infection has long been
endemic, such as Japan and Egypt, there were high prevalences of HCV infection among cirrhosis and
HCC patients. The same held true for China and most of the African nations in our sample regarding
HBV infection. Areas such as these, where HBV infection predominated, appeared to have a younger
population of HCC cases, which is thought to reflect the preponderance of infections acquired early in
life (e.g., perinatal HBV transmission) [8]. Patterns of HBV and HCV co-infection were also notable.
When we applied the HBV- and HCV-attributable fractions we derived to 2002 worldwide mortality
estimates [1], we found that approximately 929,000 deaths from cirrhosis (n = 446,000) and primary
liver cancer (n = 483,000) were likely due to chronic viral hepatitis infections. HBV infection accounted
for 563,000 deaths (235,000 from cirrhosis and 328,000 from liver cancer) and HCV infection
accounted for 366,000 deaths (211,000 from cirrhosis and 155,000 from liver cancer).
We showed that chronic viral hepatitis infections likely account for the majority of both cirrhosis and
HCC globally and in nearly all regions of the world. One of the strengths of our analysis was that it
employed simple and transparent methods. Our estimates of attributable fractions were derived from
reviews of published studies reporting the prevalence of HBV and HCV infections in patients with
cirrhosis or HCC in all regions of the world. Alternate approaches rely on estimates of the prevalence
of risk factors and corresponding relative risks in the source populations. However, errors associated
with extrapolating exposure or hazard from one population to another are a major source of
uncertainty in efforts to characterize international health risks [12]. Given the lack of representative
data regarding HBV and HCV infection prevalences worldwide along with uncertainties in deriving
region specific risk estimates, we believe ours is the preferred approach.
Our findings help illustrate the great need for programs aimed at preventing HBV or HCV transmission.
In 1992, WHO recommended that all countries include hepatitis B vaccine in their routine infant
immunization programs. As of 2003, WHO/UNICEF estimated 42% hepatitis B vaccination coverage
among the global birth cohort [106]. Therefore, implementation of this strategy, which represents the

most effective way of preventing chronic HBV infection and related end stage liver disease, is far from
complete [107,108]. Other key primary prevention strategies include screening blood donors and
maintaining infection control practices to prevent the transmission of healthcare-related HBV and HCV
infections [105,109,110]. In countries where these activities have not been fully implemented, they
should be given a high priority. In most developed countries, injection drug use and high-risk sexual
behaviors represent the major risk factors for HCV infection and HBV infection, respectively, indicating
the importance of related prevention efforts (e.g., reducing the numbers of new initiates to injection
drug use).
The role of programs to identify, counsel, and provide medical management for the many persons
already infected with HBV or HCV requires careful consideration [105,110]. Counseling that includes
advice regarding avoidance of alcohol and education regarding modes of transmission can help reduce
the risks for developing chronic disease or spreading infection to susceptible persons. The widespread
application of therapeutic interventions also has the potential to accelerate the declines in end-stage
liver disease that will eventually follow from hepatitis B vaccination and other primary prevention
efforts [104,107]. Recent advances have occurred in the therapeutic management of chronic hepatitis
B and chronic hepatitis C, but treatments are long and involve substantial costs and side effects [111
113]. Countries will need to consider the potential benefits of treatment while insuring that scarce
healthcare resources are allocated in a manner that does not undermine primary prevention efforts
[114].
Risk of pancreatic cancer among individuals with hepatitis C or hepatitis B virus infection: a
nationwide study in Sweden.
Huang J1, Magnusson M, Trner A, Ye W, Duberg AS.
Br J Cancer. 2013 Nov 26; 109(11):2917-23.
http://dx.doi.org:/10.1038/bjc.2013.689
A few studies indicated that hepatitis C and hepatitis B virus (HCV/HBV) might be associated with
pancreatic cancer risk. The aim of this nationwide cohort study was to examine this possible
association. Methods: Hepatitis C virusand hepatitis B virus-infected individuals were identified from the national surveillance database from
1990 to 2006, and followed to the end of 2008. The pancreatic cancer risk in the study population was
compared with the general population by calculation of Standardized Incidence Ratios (SIRs), and with
a matched reference population using a Cox proportional hazards regression model to calculate hazard
ratios (HRs). Results: In total 340 819 person-years in the HCV cohort and 102 295 in the HBV cohort

were accumulated, with 34 and 5 pancreatic cancers identified, respectively. The SIRHCV was 2.1
(95% confidence interval, CI: 1.4, 2.9) and the SIRHBV was 1.4 (0.5, 3.3). In the Cox model analysis,
the HR for HCV infection was 1.9 (95% CI: 1.3, 2.7), diminishing to 1.6 (1.04, 2.4) after adjustment
for potential confounders.
Conclusion: Our results indicated that HCV infection might be associated with an increased risk of
pancreatic cancer but further studies are needed to verify such association. The results in the HBV
cohort indicated an excess risk, however, without statistical significance due to lack of power.
Pancreatic cancer is one of the most rapidly fatal malignancies with a 5-year survival rate below 5%.
The long-term survival is poor also for early diagnosed patients treated with resection surgery
(Jemal et al, 2010). In Europe, it was estimated in a prediction model that in the year 2012 there
would be 7500080000 deaths from pancreatic cancer, which is the fourth most common cause of
cancer-associated death for both men and women (Malvezzi et al, 2012). The incidence of pancreatic
cancer is higher in the Nordic countries and Central Europe than in other parts of the world (Bosetti et
al, 2012).
Tobacco smoking is a well-established risk factor for pancreatic cancer (Iodice et al, 2008), and a
similar magnitude of excess risk as smoking was found among the users of Scandinavian snus (moist
snuff) (Boffettaet al, 2005; Luo et al, 2007). Besides, accumulating evidence consistently shows that
old age, male sex, diabetes mellitus, hereditary pancreatitis, chronic pancreatitis and family history
are positively associated with this carcinoma (Pandol et al, 2012). Albeit the biological mechanism is
unclear, recent epidemiological studies indicated that some infections, such as exposure
to Helicobacter pylori (Trikudanathan et al, 2011), poor oral health (Michaud et al, 2007), hepatitis C
virus (HCV) (Hassan et al, 2008; El-Serag et al, 2009) or hepatitis B virus (HBV) (Hassan et al,
2008; Iloeje et al, 2010; Wang et al, 2012a, 2012b) might be associated with pancreatic cancer risk.
Globally, 170 million people are chronically infected with HCV (World Health Organization, 1997) and
an estimated 350 million with HBV (Custer et al, 2004). The prevalence rates of HCV and HBV
infection vary widely in the world, and Sweden is a low endemic country with an estimated 0.5% of
the population infected with HCV (Duberg et al, 2008a) and even lower rate for HBV infection. Both
chronic HCV and HBV infections are main causes of hepatocellular carcinoma (HCC). Previous findings
demonstrated that HBV may replicate within the pancreas (Shimoda et al, 1981; Yoshimura et al,
1981) and that HCV could be associated with pancreatitis (Alvares-Da-Silva et al, 2000; Torbenson et
al, 2007). Some studies support that HCV and HBV may have a role in the development of pancreatic
cancer, but the evidence is far from conclusive (Hassan et al, 2008; El-Serag et al, 2009; Iloeje et al,
2010; Wang et al, 2012a, 2012b), and more studies are needed. Towards this end, we utilised

Swedish population-based nationwide registers, with documentation of all diagnosed HCV- and HBVinfected individuals in Sweden, to explore the association of HCV or HBV infection and the risk of
pancreatic cancer.
Baseline characteristics of the HCV and HBV cohorts are presented in Table 1. In the HCV and chronic
HBV cohorts the mean follow-up time were 9.1 and 9.4 years, with a total of 360154 and 107986
person-years at risk, respectively. There was a clear male dominance in the HCV cohort, and median
age at entry into the HCV or HBV cohorts (notification date) was 38 and 31 years, respectively. A
marked difference between cohorts was observed regarding the aspect of country of origin; HCVinfected individuals were more likely from Nordic countries, but persons with chronic HBV infection
were often immigrants from non-Nordic countries.
Hepatitis C virus cohort
In the HCV cohort, there were 34 pancreatic cancer cases observed during 340819 person-years of
follow-up (first 6 months of follow-up excluded), whereas 16.5 were expected, yielding a statistically
significant increased risk of pancreatic cancer (SIR: 2.1; 95% CI: 1.4, 2.9). The SIR did not alter
substantially across sex or estimated duration of HCV infection (Table 2). The majority of cases were
among the patients who were born before 1960.
From the Cox regression model, an 90% excessive risk for pancreatic cancer (HR 1.9; 95% CI: 1.3,
2.7) was observed after adjustment for age, sex and county of residence, which is similar to the result
from the SIR analysis. This excess risk diminished somewhat but remained statistically significant after
further adjustment for potential confounders (HR 1.6; 95% CI: 1.04, 2.4). The results did not vary
markedly when stratified by sex (Table 3). In the additional analyses, excluding all individuals ever
hospitalized with acute and/or chronic pancreatitis, the results did not alter notably (data not shown).
In the HCV cohort, the Standardized Incidence Ratio (SIR) for lung cancer was 2.3 (95% CI: 1.9, 2.7)
and the Hazard Ratio (HR) for lung cancer was 2.2 (95% CI: 1.8, 2.7), decreasing to 1.6 (95% CI:
1.3, 2.1) after adjustment for the potential confounders used in the pancreatic cancer analyses.
Chronic HBV cohort
A total of five pancreatic cancer cases were found during 102295 person-years of follow-up (first 6
months excluded), whereas 3.5 were expected. Compared with the age- and sex-matched Swedish
general population, a 40% excess risk of pancreatic cancer was found in the chronic HBV cohort (SIR:
1.4; 95% CI: 0.5, 3.3), but without statistical significance. Because of the small number of pancreatic
cancer cases, there was not enough power for additional stratified analyses (Table 4).

The Cox regression model revealed similar results as the SIR analysis. The point estimates were
somewhat higher (HR=2.0 from the model adjusted for only matching factors and HR=1.8 from the
fully adjusted model), but still statistically non-significant (Table 5). The SIR for lung cancer in the
chronic HBV infection cohort was 1.7 (95% CI: 1.1, 2.5).
This population-based large cohort study revealed a doubled risk of pancreatic cancer among HCVinfected patients compared with the Swedish general population. The excess risk was persistent across
strata by sex or duration of infection. Although further adjustment for potential confounders, i.e.,
chronic obstructive pulmonary disease (related to smoking), diabetes mellitus, chronic pancreatitis
and alcohol-related disease, resulted in an attenuated relative risk, this finding still supports the
hypothesis that HCV infection might be associated with an increased risk of pancreatic cancer. Besides,
the result indicated a moderate excessive risk of pancreatic cancer among HBV-infected patients
according to different statistical approaches, but the size of the study cohort and the observed number
of cancers were too small to draw a sound conclusion. Pancreatic cancer is more common in older age
groups, and the small number of pancreatic cancers among the HBV cohort was probably an effect of
the relatively young cohort, concordant with the epidemiology of chronic hepatitis B in Sweden.
The strengths of this register-based study include population-based cohort design, relatively large
sample size, independently collected data on documentation of HCV/HBV notifications and pancreatic
cancer occurrence and high completeness of follow-up.
The parallel (laboratory and clinician) notification system of HCV/HBV infections in Sweden has a high
coverage of those with a diagnosed infection; it is estimated that about 7580% of HCV infections are
diagnosed, but there still remain unknown infections, not yet diagnosed or documented. In addition, a
small portion of the reported patients could have a resolved infection, spontaneously or by treatment,
this could (probably insignificant) lower the risk in the HCV and HBV cohort.
The number of unidentified HCV/HBV-coinfected individuals is probably low in the studied cohorts.
However, in the HCV cohort there could be some patients who were never diagnosed with hepatitis B
but have serologic markers of a past HBV infection. In these patients we cannot exclude the possibility
of occult hepatitis B.
The biological mechanism of the association between HCV and pancreatic cancer is unclear. However,
virtually, the pancreas and liver share the common blood vessels and ducts, and prior evidence
demonstrated that the pancreas is a remote location for hepatitis virus inhabitation and replication
(Hassan et al, 2008). HCV infection is associated with type 2 diabetes, which is both a risk factor and

might be a consequence of pancreatic cancer (Mehta et al, 2000; Sangiorgio et al, 2000). Besides,
previous studies reported that subclinical/acute pancreatitis (Katakura et al, 2005) and hyperlipasemia
(Yoffe et al, 2003) may be extrahepatic manifestations of HCV infection. In addition, pancreatic
involvement was observed among patients who suffered from chronic hepatitis infection, resulting in
mild pancreatic damage accompanied with increased serum levels of pancreatic enzyme (Taranto et al,
1989; Katakura et al, 2005). Immune response may lead to chronic inflammation in the targeted
organs after long time persistent infection with HCV. Therefore, hepatitis C virus conceivably serves as
a biological agent that may indirectly have a role in inflammation-associated pancreatic
carcinogenesis. Although still unclear to what extent chronic inflammation contributes to pancreatic
cancer development, it is postulated that HCV can induce inflammatory microenvironment with high
concentration of growth factors and cytokines. This may exert effects by accumulating alterations in
driver genes and promoting cancer cell growth and proliferation.
HIV AIDS and Cancer
http://www.cancer.gov/cancertopics/causes-prevention/risk/infectious-agents/hiv-fact-sheet
Key Points

People infected with human immunodeficiency virus (HIV) have a higher risk of some
types of cancer than uninfected people.

A weakened immune system caused by infection with HIV, infection with other viruses,
and traditional risk factors such as smoking all contribute to this higher cancer risk.

Highly active antiretroviral therapy and lifestyle changes may reduce the risk of some
types of cancer in people infected with HIV.

The National Cancer Institute (NCI) conducts and supports a number of research
programs aimed at understanding, preventing, and treating HIV infection, acquired
immunodeficiency syndrome-related cancers, and cancer-associated viral diseases.

1.

Do people infected with human immunodeficiency virus (HIV) have an

increased risk of cancer?

Yes. People infected with HIV have a substantially higher risk of some types of cancer compared with
uninfected people of the same age (1). Three of these cancers are known as acquired
immunodeficiency syndrome (AIDS)-defining cancers or AIDS-defining malignancies: Kaposi
sarcoma, non-Hodgkin lymphoma, and cervical cancer. A diagnosis of any one of these cancers marks
the point at which HIV infection has progressed to AIDS.
People infected with HIV are several thousand times more likely than uninfected people to be
diagnosed with Kaposi sarcoma, at least 70 times more likely to be diagnosed with non-Hodgkin
lymphoma, and, among women, at least 5 times more likely to be diagnosed with cervical cancer (1).
In addition, people infected with HIV are at higher risk of several other types of cancer (1). These
other malignancies include anal, liver, and lung cancer, and Hodgkin lymphoma.

People infected with HIV are at least 25 times more likely to be diagnosed with anal cancer than
uninfected people, 5 times as likely to be diagnosed with liver cancer, 3 times as likely to be diagnosed
with lung cancer, and at least 10 times more likely to be diagnosed with Hodgkin lymphoma (1).
People infected with HIV do not have increased risks of breast, colorectal, prostate, or many other
common types of cancer (1). Screening for these cancers in HIV-infected people should follow current
guidelines for the general population
HIV and cancer of the cervix
Z.M. Chirenje
bestpracticeobgyn April 2005; 19(2):269276
http://dx.doi.org/10.1016/j.bpobgyn.2004.10.002
Cancer of the cervix is the second most common cause of cancer-related death in women worldwide,
and in some low resource countries accounts for the highest cancer mortality in women. The highest
burden of the HIV/AIDS epidemic is currently in sub-Saharan Africa, where more than half of the
people infected are women who have no access to cervical cancer screening. The association between
HIV and invasive cervical cancer is complex, with several studies now clearly demonstrating an
increased risk of pre-invasive cervical lesions among HIV-infected women. However, there have not
been significantly higher incidence rates of invasive cervical cancer associated with the HIV epidemic.
The highest numbers of HIV-infected women are in poorly-resourced countries, where the natural
progression of HIV disease in the absence of highly active antiretroviral treatment sometimes results
in deaths from opportunistic infections before the onset of invasive cervical cancer. This chapter will
discuss the association of HIV and cervical intraepithelial neoplasia, the treatment of pre-invasive
lesions, and invasive cervical cancer in HIV-infected women. The role of screening and the impact of
antiretroviral treatment on the progression of pre-invasive and invasive cancer will also be discussed.
Anal cancer: an HIV-associated cancer
Klencke BJ, Palefsky JM
Hematology/oncology Clinics of North America [2003, 17(3):859-872]
http://dx.doi.org:/1016/S0889-8588(03)00039-X
Although not yet included in the Centers for Disease Control definition of AIDS, anal cancer clearly
occurs more commonly in HIV-infected patients. An effective screening program for those groups who
are at highest risk might be expected to impact rates of anal cancer just as significantly as did cervical
Pap screening programs for the incidence of cervical cancer. Despite a relatively low rate of
progression from AIN to invasive cancer, the scope of the problem is enormous based on the

prevalence of anal HPV infection and the size of the HIV-infected, at-risk population. Thus, the
potential benefits of screening, detection, and the development of more effective therapy also are
enormous. Currently, therapeutic HPV vaccines for AIN represent an exciting avenue of research in
HPV-related anogenital disease. Invasive anal cancer and HSIL (which is believed to be the precursor
lesion) are expected to become increasingly important health problems for both HIV-infected men and
women as their life expectancy lengthens. Although HAART may have improved the ability of many to
tolerate CMT, it appears that toxicity of this therapy continues to be a problem for a proportion of HIVinfected subjects. The acute side effects present specific challenges to the clinician and patient, have
an immediate impact on the patients plan of care and dose intensity of the treatment, and ultimately
may impact the outcome of the planned treatment. Late toxicity may influence the long-term quality
of life. Small patient numbers, variable radiation therapy doses, limited information about viral load,
and a potential confounding effect of higher CD4+ levels make it difficult to draw any conclusions
about the effect of HAART on anal cancer outcome. Large, prospective studies will be required before
solid conclusions about the impact of various factors on anal cancer prognosis and outcome can be
drawn.
The therapeutic potential of CXCR4 antagonists in the treatment of HIV infection, cancer
metastasis and rheumatoid arthritis
Hirokazu Tamamura, and Nobutaka Fujii
Exp Opin on Ther Targets Dec 2005; 9(6): 1267-1282http://dx.doi.org:/10.1517/14728222.9.6.1267
CXCR4 is the receptor of the chemokine CXCL12, which is involved in progression and metastasis of
several types of cancer cells, HIV infection and rheumatoid arthritis. The authors developed selective
CXCR4 antagonists, T22 and T140, initially as anti-HIV agents, which inhibit T cell line-tropic (X4-)
HIV-1 infection through their specific binding to CXCR4. Recently, T140 analogues have also been
shown to inhibit CXCL12-induced migration of breast cancer cells, leukaemia T cells, pancreatic cancer
cells, small cell lung cancer cells, chronic lymphocytic leukaemia B cells, pre-B acute lymphoblastic
leukaemia cells and so on in vitro. Biostable T140 analogues significantly suppressed pulmonary
metastasis of breast cancer cells and melanoma cells in mice. Furthermore, these compounds
significantly suppressed the delayed-type hypersensitivity response induced by sheep red blood cells
and collagen-induced arthritis, which represent in vivo mouse models of arthritis. Thus, T140
analogues proved to be attractive lead compounds for chemotherapy of these problematic diseases.
This article reviews recent research on T140 analogues, referring to several other CXCR4 antagonists.
Types of Cancer: AIDS/HIV related malignancies

http://cancer.northwestern.edu/cancertypes/cancer_type.cfm?category=1
People with HIV/AIDS are at high risk for developing certain cancers, such as Kaposis sarcoma, nonHodgkin lymphoma, and cervical cancer. For people with HIV, these three cancers are often called
AIDS-defining conditions, meaning that if a person with HIV has one of these cancers it can signify
the development of AIDS. The connection between HIV/AIDS and certain cancers is not completely
understood, but the link likely depends on a weakened immune system. Most types of cancer begin
when normal cells begin to change and grow uncontrollably, forming a mass called a tumor. A tumor
can be benign (noncancerous) or malignant (cancerous, meaning it can spread to other parts of the
body). The types of cancer most common for people with HIV/AIDS are described in more detail
below.
Kaposis sarcoma
Kaposis sarcoma is a type of skin cancer, which has traditionally occurred in older men of Jewish or
Mediterranean descent, young men in Africa, or people who have received organ transplantation.
Today, Kaposis sarcoma is found most often in homosexual men with HIV/AIDS and related to an
infection with the human herpesvirus 8 (HHV-8). Kaposis sarcoma in people with HIV is often called
epidemic Kaposis sarcoma. HIV/AIDS-related Kaposis sarcoma causes lesions to arise in multiple sites
in the body, including the skin, lymph nodes, and organs such as the liver, spleen, lungs, and digestive
tract.
Non-Hodgkin lymphoma
HIV/AIDS-related NHL is the second most common cancer associated with HIV/AIDS, after Kaposis
sarcoma. There are many different subtypes of NHL. The most common subtypes of NHL in people
with HIV/AIDS are primary central nervous system lymphoma (affecting the brain and spinal fluid),
found in 20% of all NHL cases in people with HIV/AIDS, primary effusion lymphoma (causing fluid to
accumulate around the lungs or in the abdomen), or intermediate and high-grade lymphoma. More
than 80% of lymphomas in people with HIV/AIDS are high-grade B-cell lymphoma, while 10% to 15%
of lymphomas among people with cancer who do not have HIV/AIDS are of this type. It is estimated
that between 4% and 10% of people with HIV/AIDS develop NHL.
Other types of cancer
Other, less common types of cancer that may develop in people with HIV/AIDS are Hodgkins
lymphoma, angiosarcoma (a type of cancer that begins in the lining of the blood vessels), anal cancer,

liver cancer, mouth cancer, throat cancer, lung cancer, testicular cancer, colorectal cancer, and multiple
types of skin cancer including basal cell carcinoma, squamous cell carcinoma, and melanoma.
Treatment options for the most common treatments for HIV/AIDS-related cancers are listed by the
specific type of cancer. Treatment options and recommendations depend on several factors, including
the type and stage of cancer, possible side effects, and the patients preferences and overall health.
Palliative care can help a person at any stage of illness. People often receive treatment for the cancer
and treatment to ease side effects at the same time. In fact, patients who receive both often have less
severe symptoms, better quality of life, and report they are more satisfied with treatment.
Palliative treatments vary widely and often include medication, nutritional changes, relaxation
techniques, and other therapies. You may also receive palliative treatments similar to those meant to
eliminate the cancer, such as chemotherapy, surgery, and radiation therapy.
It is extremely important that all patients with HIV/AIDS and an associated cancer receive treatment
with highly active antiretroviral treatment (HAART) both during the cancer treatments and afterwards.
HAART can effectively control the virus in most patients. Better control of the HIV infection decreases
the side effects of many of the treatments, may decrease the chance of a recurrence, and can improve
a patients chance of recovery from the cancer.
The treatment of HIV/AIDS-related Kaposi sarcoma usually cannot cure the cancer, but it can help
relieve pain or other symptoms. This can be followed by palliative care for Kaposi sarcoma. Antiviral
treatment for HIV/AIDS helps reduce a persons chance of getting Kaposi sarcoma and can reduce the
severity of Kaposi sarcoma. HAART helps treat the tumor and reduce the symptoms associated with
Kaposi sarcoma for people with HIV/AIDS. It is usually used before other treatments, such as
chemotherapy.
Curettage and electrodesiccation. In this procedure, the cancer is removed with a curette, a sharp,
spoon-shaped instrument. The area can then be treated with electrodesiccation, which uses an electric
current to control bleeding and kill any remaining cancer cells. Many patients have a flat, pale scar
from this procedure.
Cryosurgery. Cryosurgery, also called cryotherapy or cryoablation, uses liquid nitrogen to freeze and
kill cells. The skin will later blister and shed off. This procedure will sometimes leave a pale scar. More
than one freezing may be needed.

In photodynamic therapy, a light-sensitive substance is injected into the lesion that stays longer in
cancer cells than in normal cells. A laser is directed at the lesion to destroy the cancer cells.
Radiation therapy is the use of high-energy x-rays or other particles to destroy cancer cells. A doctor
who specializes in giving radiation therapy to treat cancer is called a radiation oncologist. The most
common type of radiation treatment is called external-beam radiation therapy, which is radiation given
from a machine outside the body. When radiation therapy is given using implants, it is called internal
radiation therapy or brachytherapy. External-beam radiation therapy may be given as a palliative
treatment. A radiation therapy regimen (schedule) usually consists of a specific number of treatments
given over a set period of time.
Side effects from radiation therapy may include fatigue, mild skin reactions, upset stomach, and loose
bowel movements. Most side effects go away soon after treatment is finished. Learn more about
radiation therapy.
Chemotherapy may help control advanced disease, although curing HIV/AIDS-related Kaposi sarcoma
with chemotherapy is extremely rare. Usually, for HIV/AIDS-related Kaposi sarcoma, chemotherapy is
used to help relieve symptoms and to lengthen a patients life. Common drugs for Kaposi sarcoma
include: liposomal doxorubicin (Doxil), paclitaxel (Taxol, LEP-ETU, Abraxane), and vinorelbine
(Navelbine, Alocrest).
The side effects of chemotherapy depend on the individual and the dose used, but they can include
fatigue, risk of infection, nausea and vomiting, hair loss, loss of appetite, and diarrhea. These side
effects usually go away once treatment is finished.
HIV/AIDS-related Kaposi sarcoma may receive alpha-interferon (Roferon-A, Intron A, Alferon), which
appears to work by changing the surface proteins of cancer cells and by slowing their growth.
Immunotherapy is generally used for people who are in the good-risk category in the immune system
(I) factor of the TIS staging system (see Stages). The most common side effects of alpha-interferon
are low levels of white blood cells and flu-like symptoms.
The main treatments for HIV/AIDS-related non-Hodgkin lymphoma are chemotherapy, targeted
therapy, and radiation therapy.
Treatments for women with the precancerous condition called CIN (see

Overview) are generally not

as effective for women with HIV/AIDS because of a weakened immune system. Often, the standard
treatment for HIV/AIDS can lower the symptoms of CIN.

Women with invasive cervical cancer and HIV/AIDS that is well-controlled with medication, generally
receive the same treatments as women who do not have HIV/AIDS. Common treatment options
include surgery, radiation therapy, and chemotherapy.
Cytokines in cancer pathogenesis and cancer therapy
Glenn Dranoff
Nature Reviews Cancer Jan 2004; 4(11-22) http://dx.doi.org:/10.1038/nrc1252
The mixture of cytokines that is produced in the tumor microenvironment has an important role in
cancer pathogenesis. Cytokines that are released in response to infection, inflammation and immunity
can function to inhibit tumor development and progression. Alternatively, cancer cells can respond to
host-derived cytokines that promote growth, attenuate apoptosis and facilitate invasion and
metastasis. A more detailed understanding of cytokinetumor-cell interactions provides new
opportunities for improving cancer immunotherapy.
Dendritic Cells as Therapeutic Vaccines against Cancer
Jacques Banchereau and A. Karolina Palucka
Nature Reviews Immunology APR 2005; 5:296-306
http://cnc.cj.uc.pt/BEB/private/pdfs/2007-2008/Immunology/E/Rev_paper_E4.pdf
Mouse studies have shown that the immune system can reject tumours, and the identification of
tumor antigens that can be recognized by human T cells has facilitated the development of
immunotherapy protocols. Vaccines against cancer aim to induce tumor-specific effector T cells that
can reduce the tumor mass, as well as tumor-specific memory T cells that can control tumor relapse.
Owing to their capacity to regulate T-cell immunity, dendritic cells are increasingly used as adjuvants
for vaccination, and the immunogenicity of antigens delivered by dendritic cells has now been shown
in patients with cancer. A better understanding of how dendritic cells regulate immune responses will
allow us to better exploit these cells to induce effective anti-tumor immunity.
Vaccines against infectious agents are one success of immunology and have spared countless
individuals from diseases such as polio, measles, hepatitis B and tetanus 8. However, progress in the
development of vaccines against infectious agents has been largely empirical and not always
successful, as many infectious diseases still evade the immune system, particularly chronic infections
such as tuberculosis, malaria and HIV infection. Further progress will be made through rational design
based on our increased understanding of how the immune system works and how the induction of
protective immunity is regulated. The same principle applies to vaccines against cancer, particularly as

cancer is a chronic disease, and when it becomes clinically visible, tumor cells and their products have
already been interacting with and affecting host cells for a considerable time to ensure the survival of
the tumor. Ex vivo-generated, antigen-loaded DCs have now been used as vaccines to improve
immunity9 . Numerous studies in mice have shown that DCs loaded with tumor antigens can induce
therapeutic and protective antitumor immunity 10. The immunogenicity of antigens delivered by DCs
has been shown in patients with cancer9 or chronic HIV infection 11, thereby providing proof of principle
that using DCs as vaccines can work. Despite this, the efficacy of therapeutic vaccination against
cancer has recently been questioned12 because of the undeniably limited rate of objective tumor
regressions that has been observed in clinical studies so far. However, the question is not whether DC
vaccines work but how to orient further studies to refine the immunological and clinical parameters of
vaccination with DCs to improve its efficacy.
Vaccines against cancer Early studies in mice showed that the immune system can recognize and
reject tumours13 and that immunodeficient mice (lacking interferon- (IFN-) and recombinationactivating gene 2) have an increased incidence of cancer 14 (BOX 1). In humans, the incidence of some
cancers is increased in immunodeficient patients 15 and is increased with age, owing
toImmunosenescence16. These observations support the scientific rationale for immunotherapy for
cancer. The term immunotherapy refers to any approach that seeks to mobilize or manipulate the
immune system of a patient for therapeutic benefit17. In this regard, there are numerous strategies for
improving the resistance of a patient to cancer. These include non-specific activation of the immune
system with microbial components or cytokines, antigen-specific adoptive immunotherapy with
antibodies and/or T cells, and antigen-specific active immunotherapy (that is, vaccination). The main
limitation of using antibodies is that target proteins need to be expressed at the cell surface. By
contrast, targets for T cells are usually peptides derived from intracellular proteins, which are
presented at the cell surface in complexes with MHC molecules 18. The identification of defined tumor
antigens in humans19,20 prompted the development of adoptive T-cell therapy. Yet, the most attractive
strategy is vaccination, which is expected to induce both therapeutic T-cell immunity (in the form of
tumor-specific effector T cells) and protective T-cell immunity (in the form of tumor-specific memory T
cells that can control tumor relapse)2123. Numerous approaches for the therapeutic vaccination of
individuals who have cancer have been developed, including the use of the following: autologous and
allogeneic tumor cells (which are often modified to express various cytokines), peptides, proteins and
DNA vaccines9,2326. The observed results are variable; however, in many cases, a tumour-specific
immune response has been induced, and tumor regressions, albeit limited, have occurred. These
approaches rely on random encounter of the vaccine with host DCs. A lack of encounter of the vaccine

antigen with DCs might result in the absence of an immune response. Alternatively, an inappropriate
encounter for example, with unactivated DCs or with the wrong subset of DCs might lead to
silencing of the immune response27. Both of these situations could explain some of the shortcomings of
current cancer vaccines. Furthermore, we do not know how tumor antigens need to be delivered to
DCs in vivo to elicit an appropriate immune response.
Immature and mature dendritic cells have different functions. A | Immature dendritic cells
(DCs) induce tolerance. Tissue DCs constantly sample their environment, capture antigens and
migrate in small numbers to draining lymph nodes. In the absence of inflammation, the DCs remain in
an immature state, and antigens are presented to T cells in the lymph node without costimulation,
leading to either the deletion of T cells or the generation of inducible regulatory T cells. B | Mature DCs
induce immunity. Tissue inflammation induces the maturation of DCs and the migration of large
numbers of mature DCs to draining lymph nodes. The mature DCs express peptideMHC complexes at
the cell surface, as well as appropriate co-stimulatory molecules. This allows the priming of CD4+ T
helper cells and CD8+ cytotoxic T lymphocytes (CTLs), the activation of B cells and the initiation of an
adaptive immune response. To control the immune response, CD4+CD25+ regulatory T (TReg)-cell
populations are also expanded. [ADCC, antibody-dependent cell-mediated cytotoxicity; NK, natural
killer; TCR, T-cell receptor].
Box 1 |
Mice

The immune system can reject tumors

Immune-mediated rejection of chemically induced tumours 13

Increased cancer incidence in immunodeficient mice 14

Humans

Increased cancer incidence in immunodeficient patients 15

Increased cancer incidence with age (immunosenescence) 16

Cancer regression in patients with paraneoplastic neurological disorders that are

mediated by onconeuronal antibodies and specific CD8+ T cells 136

Dendritic cells DC subsets. There are thought to be two main pathways of differentiation into
DCs2,31 (FIG. 2). The myeloid pathway generates two subsets: Langerhans cells, which are found in
stratified epithelia such as the skin; and interstitial DCs, which are found in all other tissues 32. The
lymphoid pathway generates plasmacytoid DCs (pDCs), which secrete large amounts of type I IFNs
(IFN- and IFN-) after viral infection 33,34. DCs and their precursors show remarkable functional
plasticity. For example, pDCs form one of the first barriers to the expansion of intruding viruses,

thereby functioning, through the release of type I IFNs, as part of the innate immune response.
Subsequently, these cells differentiate into DCs that can present antigens to T cells, thereby
functioning as members of the adaptive immune system 35,36. Monocytes can differentiate into either
macrophages, which function as scavengers, or DCs that induce specific immune responses 37,38.
Different cytokines skew the in vitro differentiation of monocytes into DCs with different phenotypes
and functions (FIG. 3). So, after activation (for example, by granulocyte/ macrophage colonystimulating factor, GM-CSF), monocytes that encounter interleukin-4 (IL-4) become DCs known as IL4-DCs29,30,39. By contrast, after encounter with IFN-, tumour-necrosis factor (TNF) or IL-15, activated
monocytes differentiate into IFN--DCs4043, TNF-DCs44 or IL-15-DCs45, respectively. Whether, in vivo,
all interstitial DCs are derived from monocytes remains to be established, but myeloid DCs that are
isolated from human peripheral blood also give rise to different DC types after exposure to different
cytokines. Each of these DC subsets has both common and unique biological functions, which are
determined by a unique combination of cell-surface molecule expression and cytokine secretion. For
example, whereas IL-4-DCs are a homologous population of immature cells that is devoid of
Langerhans cells, TNFDCs are heterogeneous and include both CD1a+ Langerhans cells and CD14+
interstitial DCs44.In vitro experiments showed that Langerhans cells and interstitial DCs that were
generated from cultures of CD34+ hematopoietic progenitors differ in their capacity to activate
lymphocytes: interstitial DCs induce the differentiation of naive B cells into immunoglobulin-secreting
plasma cells4,32, whereas Langerhans cells seem to be particularly efficient activators of cytotoxic
CD8+ T cells. They also differ in their cytokine-secretion pattern (only interstitial DCs produce IL-10)
and their enzymatic activity4,32, which might be fundamental for the selection of peptides that are
presented to T cells. Indeed, different enzymes are likely to degrade a given antigen into different sets
of peptides, as has recently been shown for the HIV protein Nef 46. This then leads to different sets of
peptideMHC complexes being presented and thereby to distinct repertoires of antigen-specific T cells.
So, these unique DCs are likely to yield unique immune effectors, thereby allowing the broad immune
response that is required to combat permanently evolving microorganisms and tumors.
Distinct DC subsets induce distinct types of immune response. DCs have a crucial role in determining
the type of response that is induced. There is evidence that either polarized DCs or distinct DC subsets
might provide T cells with different signals that determine the class of immune response 31. So, in mice,
splenic CD8+ DCs prime naive CD4+ T cells to produce TH1 cytokines in a process that involves IL12, whereas splenic CD8 DCs prime naive CD4+ T cells to produce TH2 cytokines 47,48. Furthermore,
this polarization into different T-cell subsets also depends on the signal received by a DC, as shown by
the induction of IL-12 production and polarization towards TH1 cells when DCs are activated with

Escherichia coli lipopolysaccharide (LPS), but the absence of IL-12 production and polarization towards
TH2 cells when the same type of DC is exposed to LPS from Porphyromonas gingivalis

. In humans,

49

CD40 ligand (CD40L)-activated monocyte-derived DCs prime TH1-cell responses through an IL-12dependent mechanism, whereas pDCs activated with IL-3 and CD40L have been shown to secrete
negligible amounts of IL-12 and to prime TH2-cell responses 50. So, both the type of DC subset and the
activation signals to which DCs are exposed are important for polarization of T cells.
Mouse proof-of-principle in vivo studies

Ex vivo-generated, antigen-loaded dendritic cells (DCs) induce antigen-specific T-cell

immunity137

Ex vivo gene-loaded DCs can induce humoral immunity 138

Ex vivo-generated, antigen-loaded DCs induce tumor-specific immunity 139,140

Ex vivo-generated DCs are superior to other types of vaccine 141

Ex vivo-generated immature DCs induce tolerance142

Combination therapy with ex vivo-generated DCs improves vaccine efficacy 112,113

This is an important parameter in vaccination against cancer, as type 1 immunity (including IFN-
secretion) is desirable, whereas type 2 immunity (including IL-4 or IL-10 secretion) is considered
deleterious. DCs and immune tolerance. DCs can induce and maintain immune tolerance27, both
central and peripheral.
Central Tolerance depends on mature thymic DCs, which are essential for the deletion of newly
generated T cells that have a receptor that recognizes self-components51. However, central tolerance
might not be effective for all antigens. Furthermore, many self-antigens might not have access to the
thymus, and others are only expressed later in life. So, there is a requirement for Peripheral
Tolerance, which occurs in lymphoid organs and is mediated by immature DCs (FIG. 1a). Immature
DCs present tissue antigens to T cells in the absence of appropriate co-stimulation, leading to Tcell Anergy or deletion27 or to the development of IL-10-secreting Inducible Regulatory T Cells52,53.
The research groups of Nussenzweig and Steinman 54 have elegantly shown that fusion proteins
targeted to immature DCs lead to the induction of antigen-specific tolerance. By contrast, concomitant
activation of these DCs with CD40- specific antibody results in a potent immune response, because the
DCs are induced to express a large number of co-stimulatory molecules55. However, mature DCs
might also contribute to peripheral tolerance by promoting the clonal expansion of naturally occurring
CD4+CD25+ REGULATORY T (TReg) CELLS56, as discussed later. Therefore, the biology of DCs offers
several targets for the control of cellular immunity. The parameters that need to be considered include
DC-related factors, host-related factors and combining DC vaccines with other therapies.

Subsets of human dendritic cells. (Fig not shown). The population of dendritic cells (DCs) in the
peripheral blood, which can be mobilized by treatment with FLT3L (fms-related tyrosine kinase 3
ligand), contains both CD11c+ myeloid DCs and CD11c plasmacytoid DCs. So far, most studies of
DCs have been carried out with DCs generated by culturing monocytes with granulocyte/macrophage
colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4); this simple procedure yields a
homogenous population of DCs that resemble interstitial DCs, and the population is devoid of
Langerhans cells. These DCs are immature and require exogenous factors for maturation. DCs can also
be generated by culturing CD34+ haematopoietic progenitor cells (HPCs) or peripheral-blood
monocytes with GM-CSF and tumour-necrosis factor (TNF). In this way, two DC subsets can be
obtained: Langerhans cells, which might have improved efficacy for eliciting cytotoxic T lymphocytes;
and interstitial DCs, which resemble monocyte-derived DCs. Adding IL-4 to CD34+ HPC cultures in the
presence of GM-CSF and TNF inhibits the differentiation of Langerhans cells. [Green boxes indicate cell
types that can be induced by culture with GM-CSF and TNF. Yellow boxes indicate cell types that can
be induced by culture or mobilization with FLT3L].
Plasticity of monocyte-derived dendritic cells. (Fig not shown). Activated monocytes can
differentiate into different types of dendritic cell (DC) after encounter with different cytokines. These
distinct DCs will influence the differentiation of lymphocytes into immune effectors with different
functions, leading to varied immune responses. For example, interleukin-15-DCs (IL-15- DCs) are
remarkably more efficient at priming and maturation of rare antigen-specific cytotoxic T lymphocytes
(CTLs) than are IL-4-DCs. Thymic stromal lymphopoietin-DCs (TSLP-DCs) induce CD4+ T cells to
differentiate into pro-inflammatory T helper 2 (TH2) cells, which secrete large amounts of IL-13 and
tumor-necrosis factor (TNF)143, whereas interferon--DCs (IFN--DCs) induce CD4+ T cells to
differentiate into TH1 cells, which secrete IFN- and IL-10. The properties and function of TNF-DCs
remain to be determined. [FLT3L, fms-related tyrosine kinase 3 ligand; GM-CSF,
granulocyte/macrophage colony-stimulating factor].
Antigen loading. Loading MHC class I and class II molecules at the cell surface of DCs with peptides
derived from defined antigens is the most commonly used strategy for DC-based vaccination 22,87.
Although this technique is important for proof-of-principle studies, the use of peptides has limitations:
the restriction of a peptide to a given HLA type; the limited number of well-characterized TumorAssociated Antigens; the relatively rapid turnover of exogenous peptide MHC complexes, resulting
in comparatively low antigen presentation by the time that the DC arrives in the draining lymph node
after injection; and the induction of a restricted repertoire of T-cell clones, thereby limiting the ability
of the immune system to control tumor-antigen variation. Yet another level of complexity is brought

about by the use of MODIFIED HETEROCLITIC PEPTIDES. Some synthetic peptides, even those derived
from immune-dominant antigens, do not bind MHC class I molecules with high affinity, possibly
explaining their limited immunogenicity in vivo88. Therefore, the generation of peptide analogues with
increased affinity for MHC class I molecules (known as heteroclitic peptides) could be used to improve
peptide immunogenicity89,90. However, recent elegant studies in patients with malignant melanoma
show that T cells elicited in vivo by vaccination with heteroclitic MART1 (melanoma antigen recognized
by autologous T cells) or glycoprotein 100 (gp100) peptide show poor recognition of the endogenous
melanoma-derived peptide and less efficient tumor-cell lysis compared with T cells specific for the
native peptide91.
Immunoregulatory mechanisms
Naturally occurring CD4+CD25+ regulatory T cells
Cell-mediated suppression independent of interleukin-10 (IL-10) and/or transforming growth factor-
(TGF-);
clonal expansion is regulated by mature dendritic cells (DCs)
Inducible regulatory T cells
Cytokine-mediated suppression through IL-10 and/or TGF-; induction and clonal expansion is
regulated by immature DCs
Natural killer T cells
Cytokine-mediated suppression through IL-13
Vaccine-induced B cells?
Cytokine-mediated regulation through IL-4, IL-6 and IL-10; competition with DCs for antigen uptake
Tumor-specific interferon--secreting T cells?
Immunoediting and selection of escape variants (not discussed in main text)
Immune correlates of efficacy of dendritic-cell-based vaccines

Induction of broad tumour-specific T-cell immunity: T cells specific for several tumour
antigens

Induction of effector T cells: T cells with immediate capacity to recognize tumour

antigens and secrete cytokines such as tumour-necrosis factor and interferon-

Induction of memory T cells: T cells that secrete interleukin-2 and proliferate on reexposure to tumour antigen

Induction of T cells that kill tumour cells

Decreased number of T cells with regulatory function

DCs are an attractive target for therapeutic manipulation of the immune system to increase otherwise
insufficient immune responses to tumour antigens. However, the complexity of the DC system requires
rational manipulation of DCs to achieve protective or therapeutic immunity. So, further research is
needed to analyse the immune responses induced in patients by distinct ex vivo-generated DC subsets
that are activated through different pathways. The ultimate ex vivo-generated DC vaccine will be
heterogeneous and composed of several subsets, each of which will target a specific immune effector.
These ex vivo strategies should help to identify the parameters for in vivo targeting of DCs, which is
the next step in the development of DC-based vaccination. Indeed, distinct DC subsets express unique
cell-surface molecules, such as different lectins 131: Langerhans cells express langerin, which is crucial
for the formation of Birbeck granules132,133; interstitial DCs express DCSIGN (dendritic-cell-specific
intercellular-adhesionmolecule-3-grabbing non-integrin), which is involved in interactions with T cells
and in DC migration but is also used by pathogens (such as HIV) to hijack the immune system; and
pDCs express yet another lectin, BDCA2 (blood DC antigen 2) 134,135. Such differential expression of
cell-surface molecules might allow specific in vivo targeting of DC subsets for induction of the desired
type of immune response.
9.1.2

Simultaneous Humoral and Cellular Immune Response against CancerTestis Antigen

NY-ESO-1: Definition of Human Histocompatibility Leukocyte Antigen (HLA)-A2binding

Peptide Epitopes
Elke Jger, Yao-Tseng Chen, Jan W. Drijfhout, Julia Karbach, et al.
J Exp Med. 1998 Jan 19; 187(2): 265270.
A growing number of human tumor antigens have been described that can be recognized by cytotoxic
T lymphocytes (CTLs) in a major histocompatibility complex (MHC) class Irestricted fashion.
Serological screening of cDNA expression libraries, SEREX, has recently been shown to provide
another route for defining immunogenic human tumor antigens. The detection of antibody responses
against known CTL-defined tumor antigens, e.g., MAGE-1 and tyrosinase, raised the question whether
antibody and CTL responses against a defined tumor antigen can occur simultaneously in a single
patient. In this paper, we report on a melanoma patient with a high-titer antibody response against
the cancertestis antigen NY-ESO-1. Concurrently, a strong MHC class Irestricted CTL reactivity
against the autologous NY-ESO-1positive tumor cell line was found. A stable CTL line (NW38-IVS-1)
was established from this patient that reacted with autologous melanoma cells and with allogeneic
human histocompatibility leukocyte antigen (HLA)-A2, NY-ESO-1positive, but not NY-ESO-1
negative, melanoma cells. Screening of NY-ESO-1 transfectants with NW38-IVS-1 revealed NY-ESO-1
as the relevant CTL target presented by HLA-A2. Computer calculation identified 26 peptides with HLA-

A2binding motifs encoded by NY-ESO-1. Of these, three peptides were efficiently recognized by
NW38-IVS-1. Thus, we show that antigen-specific humoral and cellular immune responses against
human tumor antigens may occur simultaneously. In addition, our analysis provides a general strategy
for identifying the CTL-recognizing peptides of tumor antigens initially defined by autologous antibody.
There is growing evidence for humoral and cellular immune recognition of cancer by the autologous
human host (16). Based on CTL-dependent lysis of cultured melanoma cell lines, several categories
of autoimmunogenic tumor antigens have been characterized, including differentiation antigens of
specific cell lineages (79), individual antigens caused by point mutations (10, 11), and tumor
antigens, such as MAGE, which are expressed in a variable proportion of different tumor types, but are
silent in most normal tissues except the testis (12). CTL responses against melanoma antigens
induced by peptide vaccines in vivo have been associated with a favorable development of advanced
melanoma in some patients (6, 13). As immunoselection of antigen-negative tumor cell variants has
been observed during peptide vaccination (14), the molecular characterization of additional CTLdefined tumor antigens is needed to develop polyvalent vaccines with broader immunotherapeutic
effects.
Sahin et al. have recently introduced a powerful new methodology for identifying human tumor
antigens eliciting humoral immune response (5). The method has been called SEREX, for serological
expression cloning of recombinant cDNA libraries of human tumors. Novel and previously defined
tumor antigens have been identified by the SEREX method, including MAGE-1 and tyrosinase, both
originally identified by cloning the epitopes recognized by CTLs. Thus, antibody screening of cDNA
libraries prepared from human tumors can be used to identify antigens eliciting a cellular immune
response, including CTLs, circumventing the need for established cultured autologous cell lines and
stable CTL lines.
We have recently identified a novel human tumor antigen by SEREX analysis of a human esophageal
cancer (15). The antigen, NY-ESO-1, belongs to a growing number of human tumor antigens we have
called cancertestis antigens that include MAGE, GAGE, BAGE (1), and SSX2 (HOM-MEL-40) (5, 16).
These antigens have the following characteristics: (a) they are expressed in a variable portion of a
wide range of cancers, (b) their normal tissue expression is generally restricted to the testis, and (c)
they are generally coded for by genes on the X chromosome. In a recent survey of sera from normal
individuals and cancer patients, antibodies against NY-ESO-1 were found in 10% of patients with
melanoma, ovarian cancer, and other cancers, but not in normal individuals (Stockert, E., manuscript
in preparation). One patient with a high NY-ESO-1 antibody response was found to have specific CTL

reactivity against cultured autologous melanoma cells. In the present study, we report that NY-ESO-1
encodes the CTL target in this patient and identify the NY-ESO-1 peptides that are recognized.
High-titer Antibody Reactivity against NY-ESO-1.
Melanoma patient NW38 presented with extensive metastases to inguinal lymph nodes having large
areas of necrosis. Reverse transcriptase PCR of tumor RNA showed that this tumor expressed NY-ESO1. Based on the hypothesis that exposure of the immune system to large amounts of intracellular
tumor proteins released from the necrotic tumor might elicit a strong humoral immune response, the
serum of patient NW38 was tested for specific reactivity against recombinant NY-ESO-1 protein. Fig.
Fig.11 shows the reactivity of NW38 serum with the recombinant NY-ESO-1 protein, with a lysate of
NY-ESO-1transfected COS-7 cells, and with a lysate of the autologous NY-ESO-1 messenger RNA
positive tumor cell line NW-MEL-38. A 22-kD protein species was identified in both cell lysates, and
comigrated with the purified recombinant NY-ESO-1 protein. The identity of this protein species as NYESO-1 was further confirmed by using an antiNY-ESO-1 mouse monoclonal antibody. Reactivity
against recombinant NY-ESO-1 protein was still detectable at a serum dilution of 1:100,000. No
reactivity was detected against a lysate of untransfected COS-7 cells.
The correlation between NY-ESO-1 expression and NW38-IVS-1 reactivity suggested NY-ESO-1 as the
antigenic target. To prove this, COS-7 cells were transfected with NY-ESO-1 cDNA and different MHC
class I molecules and used as targets for NW38-IVS-1. Reactivity was measured in a standard TNF-
release assay. TNF release was found after stimulation of NW38-IVS-1 with COS-7 cells cotransfected
with HLA-A2 and NY-ESO-1 cDNA. No reactivity was detected after stimulation with cotransfectants of
pcDNA3.1()-NY-ESO-1 and pcDNA1Amp-HLA-A1 cDNA, COS-7 cells transfected with pcDNA3.1(),
or untransfected COS-7 cells (Fig. (Fig.3).3).
Peptide-specific CTLs.
26 different peptides encoded by NY-ESO-1 with theoretical binding motifs to the HLA-A2.1 molecule
were tested for specific recognition by NW38-IVS-1. The target cells were peptide-pulsed T2 cells. Of
these 26 peptides, three were recognized by NW38-IVS-1 as determined by a standard 51Crrelease
assay (Table (Table1).1). The peptide sequences SLLMWITQCFL, SLLMWITQC, and QLSLLMWIT are
located between positions 155 and 167 of the NY-ESO-1 protein (15), and show overlapping
sequences. The 11-mer SLLMWITQCFL (2 in Table Table1)1) and the 9-mer SLLMWITQC (12 in Table
Table1)1) consist of identical amino acids at positions 19.
To provide additional confirmation of the peptide specificity, the 26 synthetic peptides were individually
incubated with HLA-A2transfected COS-7 cells and tested in the TNF release assay. Consistent with
the results of

Crrelease assay, specific TNF- release was detected in tests with peptides

51

SLLMWITQCFL, SLLMWITQC, and QLSLLMWIT. NY-ESO-1/HLA-A2 transfectants were used as a positive

control in these assays (Fig. (Fig.4).4).
The search for tumor antigens that induce specific immune responses in cancer patients is the ongoing
challenge in tumor immunology. Evidence for a specific humoral response to human cancer came from
serological analysis of cell surface reactivity of sera from cancer patients for autologous cancer cells,
an approach called autologous typing (4). However, with only a few exceptions, this approach did not
allow for the structural definition of the antigenic target. An autologous typing system also provided
the first evidence for the development of CTLs with specificity for human melanoma cells (3, 17, 21
24). Using specific antitumor CTLs as probes, a number of CTL targets have been cloned on the basis
of MHC class Irestricted recognition (1, 6). However, this approach involves cultured cancer cell lines
and stable CTL lines from the same patient, two requirements that cannot easily be met with many
tumor types. With the demonstration that genes coding for CTL-recognized tumor antigens elicit
humoral immunity and can be cloned by SEREX methodology, a technically less demanding approach
defining immunogenic tumor antigens is now available, one that extends the range of analysis to
tumor types that are not easily adaptable to in vitro growth and are not sensitive targets for CTLs. A
number of novel tumor antigens have been defined by SEREX, including two new members of the
cancertestis antigenic family, SSX2 (HOM-MEL-40) (5, 16), and NY-ESO-1 (15).
In this study, we identified a melanoma patient, NW38, with high-titered antibody against NY-ESO-1.
This patient had a large and highly necrotic tumor, and the sustained release of intracellular antigens
that are usually inaccessible to the immune system may account for the high NY-ESO-1 titer. The
establishment of an autologous cell line that typed NY-ESO-1 positive provided target cells for
assessing CTL reactivity in this patient. A CTL line was established from this patient that lysed the
autologous melanoma cell line in an HLA-A2restricted fashion. Using target cells transfected with NYESO-1 and HLA-A2, the specificity of CTL reactivity was found to be coded by NY-ESO-1. Computer
analysis of the NY-ESO-1 sequence identified 26 peptides with HLA-A2binding motifs. Screening of
these peptides presented by T2 cells identified three sequences that were confirmed to be specifically
recognized by NW38-IVS-1. This is the first conclusive demonstration of simultaneous antibody and
CTL responses against a cancertestis antigen in a single patient.
The strategy used in this study to generate and analyze CTL reactivity to a SEREX-defined antigen can
be used as a model for investigating cellular immune responses to the growing list of other SEREX
antigens. Identification of clones in SEREX requires high-titered IgG antibody, and the development of
such antibodies requires the help of CD4+ T cells. In this sense, SEREX can be thought of as a method

to define the CD4+ T cell repertoire to human tumor antigens. Also, the presence of both NY-ESO-1
antibody and CTLs in patient NW38 suggests that screening for an antibody response may be a simple
and effective way to identify patients with concomitant CTL reactivity, and this possibility is now being
tested in other patients with NY-ESO-1 antibody. In the absence of autologous tumor cell lines, CD8 + T
cells can be stimulated with autologous antigen-presenting cells that have been transfected with the
coding gene or fed purified protein antigens. A similar strategy can be used to identify peptide targets
for CD4+ T cells.
A major objective in defining immunogenic human tumor targets is to explore their use in the
development of cancer vaccines, and a number of clinical trials with various vaccine constructs are
currently underway. Although tumor regression is the desired goal of a therapeutic vaccine, this end
point cannot be expected to be an effective way to develop maximally immunogenic tumor vaccines.
For this purpose, reliable immunological assays are needed to monitor the specificity and strength of
specific immune reactions generated by the vaccine. With the exception of vaccines aimed at inducing
a humoral immune response such as GM2 ganglioside vaccines, most vaccine trials are designed to
stimulate cellular immunity, particularly the development of CTLs and CD4 + T cells. These have been
difficult to detect in vaccine trials with MAGE peptides (25), and difficult to interpret in trials with
vaccines containing melanocyte differentiation antigens, since CTLs against these antigens can be
generated in vitro from nonvaccinated melanoma patients as well as normal individuals (26, 27).
However, de novo induction and increase of preexisting CTL reactivity have been detected after
vaccination with melanocyte differentiation antigens and observed to be associated with cancer
regressions in a limited number of patients (13). The demonstration of a simultaneous antibody and
CTL response to NY-ESO-1 in the same patient suggests that serological methods may be useful in
monitoring vaccine trials with NY-ESO-1 and other tumor antigens eliciting a humoral immune
response.
9.1.3 Monoclonal Antibodies in Cancer Therapy
R K Oldham
JCO September 1983; 1(9): 582-590
http://jco.ascopubs.org/content/1/9/582.short

The need for improved specificity in cancer therapy is apparent. With the advent of monoclonal
antibodies, the possibility of specifically targeted therapy is being considered. Early trials of
monoclonal antibody in experimental animals and humans have indicated its ability to traffic to specific
tumor sites and to localize on or around the tumor cells displaying antigens to which the antibody is

directed. This evidence of specific targeting, along with preliminary evidence of therapeutic efficacy for
monoclonal antibodies and immunoconjugates with drugs, toxins, and isotopes is encouraging. The
current status of clinical trials with monoclonal antibodies is reviewed and an example of the
experimental approach for the development of immunoconjugates in animal models is presented.
Monoclonal Antibodies in Cancer Therapy: 25 Years of Progress
Robert K. Oldham, Robert O. Dillman
JCO Apr 10, 2008: 26(11): 1774-1777
http://dx.doi.org:/10.1200/JCO.2007.15.7438
In 1983, it was apparent that a major problem with current modalities of cancer treatment was the
lack of specificity for the cancer cell.1 It was predicted that a major advancement in treatment of
cancer would be the development of a class of agents that would have a greater degree of specificity
for the tumor cell. Based on many animal studies and the treatment of fewer than 100 patients, it was
evident in 1983 that monoclonal antibodies would be that major advance.
The first patient treated in the United States with monoclonal antibody therapy was a patient with
non-Hodgkins lymphoma.2 Nadler et al2 described the treatment using a murine monoclonal antibody
designated AB 89. Although treatment was not successful in inducing a significant clinical response, it
did represent the first proof of principle in humans that a monoclonal antibody could induce transient
decreases in the number of circulating tumor cells, induce circulating dead cells, and form complexes
with circulating antigen, all with minimal toxicity to the patient. Antibody could be detected on the
surface of circulating lymphoma cells, and free antigen in the serum decreased with each infusion of
antibody. After two courses of milligram doses of AB 89, a final and third course with 1.5 g of antibody
was administered during a 6-hour period. A marked reduction in circulating antigen was noted, but
these studies suggested to the authors that the quantity of circulating antigen was too great to
effectively deliver AB 89 to the patients tumor cells in a therapeutically effective manner.2
In the Journal of Clinical Oncology review article cited earlier,1 evidence was reviewed from animal
tumor models that clearly demonstrated both specificity and therapeutic efficacy with little serious
toxicity. Whereas passive serotherapy of human cancer had shown little success, 3 it was apparent in
the earlier review that monoclonal antibodies could be used in the treatment of leukemia and
lymphoma.4,5 In 1983, a review of the literature revealed approximately 10 published studies and one
in-press article of therapeutic trials of monoclonal antibody therapy in humans. All of these studies
used murine monoclonal antibodies and were phase I/II studies. Most were in leukemia or lymphoma,
but the earliest solid tumor studies were also underway in melanoma 6 and GI cancer.1

By 1983, the pioneers in monoclonal antibody research believed that a new era of cancer therapy had
begun, and for the first time, true specific and targeted therapy was underway using hybridoma
technology to produce monoclonal antibodies with exquisite specificity. It was also apparent, based on
animal model studies, that monoclonal antibodies could be a vehicle to bring immunoconjugate
therapy to the clinic by conjugating monoclonal antibodies to drugs, toxins, and radioisotopes using
the specificity of the monoclonal antibody to carry enhanced killing capacity directly to the tumor cells.
Thus, the era of monoclonal antibody therapy, as well as immunoconjugate therapy, had begun.
Although there was much excitement (and skepticism) about this new treatment modality (the use of
a form of biologic therapy with great specificity in patients with advanced cancer) there were also
problems and limitations. As presented in Table 1, there were clinical toxicities with murine monoclonal
antibodies, most of which were secondary to the interaction with the target antigen. 7 However, the
major limitation was their immunogenicity. Murine proteins are highly immunogenic, and it was soon
found that only a few infusions of these foreign proteins could be given to patients with cancer
because of the development of human antimouse antibody.8 Another problem quickly became
apparent, in that some of the antigens on cancer cell surfaces modulated off the surface and into the
circulation when antibody attached. Modulation could also cause internalization of the complex. It was
recognized that this could represent a therapeutic advantage by using the antibody as carrier to
internalize the toxic component of an immunoconjugate, potentially making it more therapeutically
active.
In 1983, few specific antigens found only in cancer cells had been identified, and there was much
debate about the specificity of these antigens. Many of the antigens to which monoclonal antibodies
were made were embryonic antigens or shared antigens found on cancer cells and some normal cells.
Therefore, although the specificity of the antibody was exquisite for the antigen, the specificity for the
antibody or immunoconjugate for cancer was not absolute. One fairly clear exception occurred early in
the 1980s when Levy et al9 developed monoclonal antibodies to the idiotype of B-lymphoma cells. The
first patient given this anti-idiotypic antibody had a complete response to therapy, and his lymphoma
went into a sustained remission that lasted for years. As a direct result of these early studies with
anti-idiotypic antibodies, there is now a series of idiotype vaccines that are in phase III trials in
patients with low-grade follicular lymphomas.10 These anti-idiotype vaccines will likely be the first truly
custom-tailored, personalized anticancer vaccines to be approved for therapeutic use.
The major limitation of murine monoclonal antibody therapy was the immunogenicity of the mouse
protein; a variety of investigators postulated that for monoclonal antibody therapy to be truly
successful, human or humanized antibodies would be necessary. It was also known 25 years ago that

the half-life of murine antibodies in the circulation was brief, and because of human antimouse
antibody, became briefer with each infusion of murine monoclonal antibody. Previous studies of human
immunoglobulin in clinical trials had demonstrated a much longer half-life for human immunoglobulin,
which predicted that once human or humanized antibodies were available, the therapeutic efficacy of
monoclonal antibodies and their immunoconjugates might be considerably enhanced. 1
How has the field of monoclonal antibody and immunoconjugate therapy fared since the predictions of
the early 1980s? Twenty-five years later, considerable progress has been made in this field. 11,12 The US
Food and Drug Administration has approved 21 monoclonal antibody products, with six of these
biologic drugs approved specifically for cancer (Table 2). It was a landmark date in November 1997
when rituximab became the first monoclonal antibody approved specifically for cancer therapy.13 In
addition to these six unconjugated monoclonal antibody therapies, one drug immunoconjugate,
gemtuzumab ozogamicin (Mylotarg; Wyeth-Ayerst, Madison, NJ), has been approved. This humanized
monoclonal antibody to CD33 is approved for use in acute myelogenous leukemia and uses the
antibody conjugated to calicheamicin, a potent enediyene antibiotic originally isolated from
aMicromonospora echoinospora.14 Two radioisotope-antibody conjugates, ytrrium-90 ibritumomab
tiuxetan (Zevalin; Cell Therapeutics Inc, Seattle, WA) and iodine-131 tositumomab (Bexxar;
GlaxoSmithKline, Middlesex, United Kingdom) have been approved. 15 The murine form of these
antibodies was retained in order to expedite clearance from the circulation. Both radiolabeled
antibodies target the CD20 antigen on lymphoma cells.
Unlike the immunoconjugates, which are currently infrequently used, each of the six unconjugated
antibodies approved for cancer therapy is currently frequently used in the treatment of humans with
cancer. The use of techniques to humanize or chimarize monoclonal antibodies to decrease their
murine components has been an important advance in the field. These molecules have a long half-life
in the blood stream, and can interact with human complement or effector cells of the patients immune
system. They behave in a manner similar to naturally occurring immunoglobulin and work along the
lines of our normal antibody-based immune response as effective agents in treating patients with
cancer.16
Rituximab has become the largest-selling biologic drug in clinical oncology, and is active in a variety of
human lymphomas and chronic lymphocytic leukemia.17,18 This is a chimeric monoclonal antibody
targeting the CD20 antigen found on both normal B cells and on most low-grade and some higher
grade B-cell lymphomas. It is effective as a single agent in induction and maintenance therapy. It is

primarily used, however, in combination with standard chemotherapies in the treatment of patients
with non-Hodgkins B-cell lymphomas and chronic lymphocytic leukemia.19-22
A second monoclonal antibody that has proven highly effective in the clinic is trastuzumab, a
humanized antibody that reacts with the second part of the human epidermal growth factor receptor
2.23 Like rituximab, it is effective as a single agent in induction and maintenance therapy, but is used
primarily in conjunction with chemotherapy for patients with human epidermal growth factor receptor
2/neupositive breast cancer.24,25
Alemtuzumab is a humanized monoclonal antibody targeting the CD52 antigen found on B
lymphocytes and is used primarily for chronic lymphocytic leukemia.26 Like the two previously cited
monoclonal antibody therapies, alemtuzumab is effective as induction and maintenance therapy.
Alemtuzumab is also reactive with T lymphocytes, and unlike the other two antibodies, it is typically
not combined with chemotherapy because of the increased risk of infection.(26)
Another humanized monoclonal antibody, bevacizumab, has been applied more broadly in human solid
tumors because it targets vascular endothelial growth factor, which is the ligand for a receptor found
on blood vessels.(27) Because this receptor is on endothelial cells, bevacizumab seems to be effective
by reducing the blood supply to tumor nodules, thereby slowing or interrupting growth. Initially
approved for advanced colorectal cancer,(28) it is now used in a variety of human solid tumors
including cancers of the lung, kidney, and breast.(29-31)
The last two antibodies approved for clinical use were cetuximab (a chimeric antibody), and
panitumumab (a completely human antibody). Both target the epidermal growth factor receptors
found on a variety of human tumors.(32,33) Cetuximab was originally approved for use in combination
with chemotherapy in metastatic colorectal cancer.(34) It also enhances chemotherapy and radiation
therapy of squamous cell cancers of the head and neck.(35) Panitumumab was approved based on its
single-agent activity in refractory colorectal cancer and is being combined with chemotherapy as well.
At the end of 2007, 25 years of clinical studies have resulted in the approval of six unconjugated,
humanized, or chimeric monoclonal antibodies for cancer therapy along with one drug
immunoconjugate and two radioisotope immunoconjugates. Although few in number, these
monoclonal antibodies are changing the face of cancer therapy, bringing us closer to more specific and
more effective biologic therapy of cancer as opposed to nonspecific cytotoxic chemicals.
Modern recombinant techniques have made it possible to rapidly produce both chimeric antibodies and
humanized antibodies, and totally human antibodies are also being produced. Identification of surface

receptors that are integral to proliferation and apoptosis has provided more targets for monoclonal
antibodies beyond those originally identified by the murine immune system. In 2008, there are more
than 100 monoclonal antibodybased biologic drugs in hundreds of clinical trials. Many of these are in
phase II and phase III and will be coming before the US Food and Drug Administration for approval in
the next few months and years. At long last, immunoconjugates are proving efficacious with
acceptable toxicity and will extend our diagnostic (36) and therapeutic armamentarium (37) from
mainly unconjugated monoclonal antibodies to a broad array of highly active and specific
immunoconjugates.
On this silver anniversary for our 1983 review, Monoclonal Antibodies in Cancer Therapy, we can
confidently predict that progress toward more specific and less toxic therapy for human cancer is in
our near future. The developments during the past 25 years in both biologic drugs and targeted small
molecules place us on the verge of more cures with less toxicity for our patients with cancer.
9.1.4 Aptamers
Nanocarriers as an emerging platform for cancer therapy
Dan Peer1,7, Jeffrey M. Karp2,3,7, Seungpyo Hong, et al.
Nature Nanotechnology 2, 751 760 (2007)
http://dx.doi.org:/10.1038/nnano.2007.387
Nanotechnology has the potential to revolutionize cancer diagnosis and therapy. Advances in protein
engineering and materials science have contributed to novel nanoscale targeting approaches that may
bring new hope to cancer patients. Several therapeutic nanocarriers have been approved for clinical
use. However, to date, there are only a few clinically approved nanocarriers that incorporate molecules
to selectively bind and target cancer cells. This review examines some of the approved formulations
and discusses the challenges in translating basic research to the clinic. We detail the arsenal of
nanocarriers and molecules available for selective tumor targeting, and emphasize the challenges in
cancer treatment.
Quantum DotAptamer Conjugates for Synchronous Cancer Imaging, Therapy, and Sensing
of Drug Delivery Based on Bi-Fluorescence Resonance Energy Transfer
Vaishali Bagalkot, L Zhang, E Levy-Nissenbaum, S Jon, PW Kantoff, et al.
Nano Letters 2007; 7(10):3065-3070
http://dx.doi.org:/10.1021/nl071546n

We report a novel quantum dot (QD)aptamer(Apt)doxorubicin (Dox) conjugate [QDApt(Dox)] as a

targeted cancer imaging, therapy, and
sensing system. By functionalizing the surface of fluorescent QD with the A10 RNA aptamer, which
recognizes the extracellular domain of the prostate specific membrane antigen (PSMA), we developed
a targeted QD imaging system (QDApt) that is capable of differential uptake and imaging of prostate
cancer cells that express the PSMA protein. The intercalation of Dox, a widely used antineoplastic
anthracycline drug with fluorescent properties, in the double-stranded stem of the A10 aptamer results
in a targeted QDApt(Dox) conjugate with reversible self-quenching properties based on a Bi-FRET
mechanism. A donoracceptor model fluorescence resonance energy transfer (FRET) between QD and
Dox and a donorquencher model FRET between Dox and aptamer result when Dox intercalated
within the A10 aptamer. This simple multifunctional nanoparticle system can deliver Dox to the
targeted prostate cancer cells and sense the delivery of Dox by activating the fluorescence of QD,
which concurrently images the cancer cells. We demonstrate the specificity and sensitivity of this
nanoparticle conjugate as a cancer imaging, therapy and sensing system in vitro.
Semiconductor nanocrystals known as quantum dots (QDs)
have been increasingly utilized as biological imaging and labeling probes because of their unique
optical properties, including broad absorption with narrow photoluminescence spectra, high quantum
yield, low photobleaching, and resistance to chemical degradation. In some cases, these unique
properties have conferred advantages over traditional fluorophores such as organic dyes. 1-4 The
surface modification of QDs with antibodies, aptamers, peptides, or small
molecules that bind to antigens present on the target cells or tissues has resulted in the development
of sensitive and specific targeted imaging and diagnostic modalities for in vitro and in vivo
applications.5-7 More recently, QDs have been engineered to carry distinct classes of therapeutic agents
for simultaneous imaging and therapeutic applications. 8,9 While these combined imaging therapy
nanoparticles represent an exciting advance in the field of nanomedicine, it would be ideal to engineer
smart multifunctional nanoparticles that are capable of performing these tasks while sensing the
delivery of drugs in a simple and easily detectable manner. One way to achieve this goal is to develop
multifunctional nanoparticles capable of sensing the release of the therapeutic modality by a change in
the fluorescence of the imaging modality.
Figure 1. (a) Schematic illustration of QD-Apt(Dox) Bi-FRET system. In the first step, the CdSe/ZnS
core-shell QD are surface functionalized with the A10 PSMA aptamer. The intercalation of Dox within
the A10 PSMA aptamer on the surface of QDs results in the formation of the QD-Apt(Dox) and

quenching of both QD and Dox fluorescence through a Bi-FRET mechanism: the fluorescence of the QD
is quenched by Dox while simultaneously the fluorescence of Dox is quenched by intercalation within
the A10 PSMA aptamer resulting in the OFF state. (b)
Schematic illustration of specific uptake of QD-Apt(Dox) conjugates into target cancer cell through
PSMA mediate endocytosis. The release of Dox from the QD-Apt(Dox) conjugates induces the recovery
of fluorescence from both QD and Dox (ON state), thereby sensing the intracellular delivery of Dox
and enabling the synchronous fluorescent localization and killing of cancer cells.
Figure 3. Fluorescence spectra. (a) QD-Apt conjugate (1 M) with increasing molar ratio of Dox (from
top to bottom: 0, 0.1, 0.3, 0.6, 1, 1.5, 2.1, 2.8, 3.5, 4.5, 5.5, 7, and 8) at an excitation of 350 nm.
(b) Dox (10 M) with increasing molar ratio of QD-Apt conjugate (from top to bottom: 0.02, 0.04,
0.07, 0.09, 0.12, 0.14, and 0.16) at an excitation of 480 nm.
In conclusion, herein we report to our knowledge the first example of a multifunctional nanoparticle
that can detect cancer cells at a single cell level while intracellularly releasing a cytotoxic dose of a
therapeutic agent in a reportable manner. We demonstrate the specificity and sensitivity of this cancer
imaging, therapy and sensing nanoparticle conjugate system in vitro by using PCa cell lines. By
functionalizing the surface of fluorescent QD with the A10 PSMA aptamer, and intercalating Dox into
the double-stranded CG sequence of the A10 PSMA aptamer, we developed a targeted QD-Apt(Dox)
conjugate with reversible Bi-FRET properties. The incorporation of multiple CG sequences within the
stem of the aptamers may further increase the loading efficiency of Dox on these conjugates. The
presence of additional Dox may enhance the selfquenching effect of QD-Apt(Dox) conjugates thereby
improving their imaging sensitivity, while the higher dose of Dox may enhance the therapeutic efficacy
of the conjugates. Furthermore, through the use of other disease-specific aptamers or other targeting
molecules, similar multifunctional nanoparticles may potentially be developed for additional important
medical applications
Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy
Hongguang Sun1, Xun Zhu2, Patrick Y Lu3, Roberto R Rosato, et al.
Molecular Therapy Nucleic Acids(2014) 3, e182;
http://dx.doi.org:/10.1038/mtna.2014.32
Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA
oligonucleotides and have high specificity and affinity for their targets. Similar to antibodies, aptamers
interact with their targets by recognizing a specific three-dimensional structure and are thus termed

chemical antibodies. In contrast to protein antibodies, aptamers offer unique chemical and biological
characteristics based on their oligonucleotide properties. Hence, they are more suitable for the
development of novel clinical applications. Aptamer technology has been widely investigated in various
biomedical fields for biomarker discovery, in vitro diagnosis, in vivo imaging, and targeted therapy.
This review will discuss the potential applications of aptamer technology as a new tool for targeted
cancer therapy with emphasis on the development of aptamers that are able to specifically target cell
surface biomarkers. Additionally, we will describe several approaches for the use of aptamers in
targeted therapeutics, including aptamer-drug conjugation, aptamer-nanoparticle conjugation,
aptamer-mediated targeted gene therapy, aptamer-mediated immunotherapy, and aptamer-mediated
biotherapy.
The terms aptamer and SELEX were introduced by two independent groups in 1990. 1,2 The term
aptamer refers to small nucleic acid ligands that exhibit specific therapeutic functions and an
unambiguous binding affinity for their targets. Conversely, Systematic Evolution of Ligands by
EXponential enrichment (SELEX) technology is the method used for aptamer development. Although
using small molecule nucleic acids as therapeutics has been explored for decades, development of
SELEX and aptamer technology revolutionized this field.
The most important property of an aptamer, from the Latin aptus (to fit), is its high target selectivity.
These short, chemically synthesized, single-stranded (ss) RNA or DNA oligonucleotides fold into
specific three-dimensional (3D) structures with dissociation constants usually in the pico- to nanomolar range.3 Moreover, in contrast to other nucleic acid molecular probes, aptamers interact with and
bind to their targets through structural recognition (Figure 1), a process similar to that of an antigenantibody reaction. Thus, aptamers are also referred to as chemical antibodies.
Figure 1.
Schematic diagram of aptamer binding to its target.
Full figure (43K)
Due to their small size and oligonucleotide properties, aptamers offer several advantages over protein
antibodies in both their extensive clinical applicability and a less challenging industrial synthesis
process. Specifically, (i) aptamers can penetrate tissues faster and more efficiently due to their
significantly lower molecular weight (825kDa aptamers versus ~150kDa of antibodies). Therefore,
aptamers penetrate tissues barriers and reach their target sites in vivo more efficiently than the
larger-sized protein antibodies. (ii) Aptamers are virtually nonimmunogenic in vivo. In principal, as
aptamers are oligonucleotides they should not be recognized by the immune system. In practice, a
recent clinical study showed that aptamers did not stimulate an immune response in vivo,4,5 as

compared to protein antibodies that are highly immunogenic, especially following repeat injections.
(iii) Aptamers are thermally stable. Based on the intrinsic property of oligonucleotides, even after a 95
C denaturation, aptamers can refold into their correct 3D conformations once cooled to room
temperature. In comparison, protein-based antibodies permanently lose their activity at high
temperatures. More importantly, a well-established synthesis protocol and chemical modification
technology lead to (iv) rapid, large-scale aptamer synthesis and modification capacity that includes a
variety of functional moieties; (v) low structural variation during chemical synthesis; and (vi) have
lower production costs. Moreover, aptamers specifically recognize a wide range of targets, such as
ions, drugs, toxins, peptides, proteins, viruses, bacteria, cells, and even tissues. 6,7,8,9,10,11,12 In the
clinic, aptamer-based therapeutics are gaining momentum. For example, Macugen, a modified RNA
aptamer, specifically targets vascular endothelial growth factor. It has been approved by the US Food
and Drug Administration (FDA)13 for the treatment of wet age-related macular degeneration and is
under evaluation for other conditions.14 In the cancer setting, AS1411 targets nucleolin, a protein
over-expressed in a variety of tumors. It is currently being evaluated as a potential treatment option
in solid tumors and acute myeloid leukemia. 15 An updated list of therapeutic aptamers undergoing
clinical trials is included in ref. 16 and Table 1. Taken together, these clinical studies highlight many
possible uses that aptamers may have in a variety of biomedical fields, including therapeutics. 17
Table 1 A list of therapeutic aptamers undergoing clinical trials.
Since aptamer technology was first introduced, the RNA-based sequence library has been widely used
for SELEX. Based on the existing evidence, it is believed that the presence of a 2-OH group and nonWatson-Crick base pairing allows RNA aptamer oligonucleotides to fold into more diverse 3D structures
than ssDNA molecules. Consequently, using the more flexible RNA sequences simplifies the
development of high-affinity and -specificity aptamers. Despite their advantages, RNA sequences are
very sensitive to nucleases present in biological environments and can be rapidly degraded. 18 To
increase nuclease resistance of RNA-based aptamers, several chemical modifications have been
investigated. Evidence shows that 2-OH group and phosphodiester linkages of RNA sequences are the
sites of nuclease hydrolysis. Subsequently, substitutions of the 2-OH functional group by 2-fluoro, 2amino, or 2-O-methoxy motifs, and/or changes to the phosphodiester backbone with
boranophosphate or phosphorothioate are the most common modifications aimed at increasing
nuclease resistance.19 More recently, Wu et al. developed a novel chemical modification method to
increase siRNA stability, in which phosphorodithioate and 2-O-Methyl were simultaneously substituted
in the same nucleotide.20
This modification method significantly enhanced siRNA stability and represents a potential new

direction for utilization of RNA-based therapies in complex biological systems. Other effective
modifications recently reported utilize the locked nucleic acid technology 16,21 or generate mirror RNA
sequence structures, termed spiegelmers. 22 These modifications result in structural changes to the
RNA sequences, which cannot be digested by nucleases.
In addition to RNA aptamers, ssDNA-based aptamers have also been developed. Due to their lack of
2-OH groups, DNA molecules are naturally resistant to 2-endonucleases and are stable in biological
environments. Recently, our group developed a biostable DNA-based aptamer specific for CD30, a
protein biomarker that is over-expressed in Hodgkin and anaplastic large cell lymphomas. Functional
analysis demonstrated that this ssDNA-based aptamer exhibited high CD30 binding affinity as low as 2
nmol/l and was stable in human serum for up to 8 hours. Conversely, an RNA-based CD30 aptamer
was digested within 10 minutes under similar conditions. 23
In summary, unique chemical features and biological functions have made aptamers a very attractive
tool in biomedical research over the past two decades. Currently, there are over 4,000 published
articles referenced in the PubMed database that include the term aptamer. Research areas that
include aptamer technology cover bioassays, drug development, cell detection, tissue staining, in
vitro and in vivo imaging, nanotechnology, and targeted therapy. As chemical antibodies, aptamers
represent an excellent alternative to replace or supplement protein antibodies, which have been
extensively used in the clinic.
Aptamers Specifically Targeting Cell Surface Biomarkers
Using SELEX technology to develop aptamers for cell surface biomarkers
SELEX, the methodology used to develop aptamers specific for a target of interest, is based on a
repetitive amplification and enrichment process. The SELEX process follows several steps: first, a
random ssDNA oligonucleotide library is chemically synthesized to contain between 10 141015 unique
random sequences flanked by conserved primer binding sites. This step utilizes the following universal
scheme: 5-sense primer sequence-(random sequence)-antisense primer sequence-3, where the
primer sequence ranges from 18 to 22 bases and the random sequence contains 2040 nucleic acids.
The general procedure consists of labeling the 5-sense primer with a fluorochrome reporter for
monitoring aptamer selection, while the 3-antisense primer is labeled with an affinity molecule, such
as biotin, that is used to separate single-stranded oligonucleotides generated in each amplification
round. This random ssDNA library can be used directly to select an initial pool of DNA aptamers.
Conversely, generation of RNA aptamers requires two extra steps. Specifically, a pool of random
ssDNA oligonucleotides is generated, T7 RNA polymerase promoter sequence is added to the 5-sense
primer, and the DNA is then used as a template for T7 RNA polymerase-based transcription in the 5 to

3 direction. During the second SELEX step, the oligonucleotide library is heated and rapidly cooled to
promote the formation of 3D structures. The library is then mixed with the target of interest for
specific binding enrichment. In the third step, the unbound sequences are discarded through the use
of membranes, columns, magnetic beads, and capillary electrophoresis. 6,24,25 In the fourth step, the
enriched sequences are amplified in vitro by either PCR (DNA aptamers) or RT-PCR (RNA aptamers) to
generate a new sequence library for the next round of SELEX. The amplified sequence library may go
through further negative-target selection, which eliminates the nonspecific sequences generated by
binding of nontarget moieties. Lastly, aptamer selection goes through 420 rounds of amplification
and enrichment. The exact number of required amplification and selection steps depends on the
aptamer target being a purified protein or a living cell, and on the evolution of the aptamer sequence
library, as that established by gel electrophoresis, flow cytometry (for target binding), classical cloning
or sequencing methods, or by high throughput Next-Generation Sequencing (NGS). In recent years,
the traditional SELEX method had also been modified to include the capillary electrophoresis (CE)
SELEX, toggle selection, photo-SELEX, bead-based selection, X-Aptamers, and Slow Off-rate Modified
Aptamers (SOMAmers) in order to maximize affinity and specificity, to improve the speed of selection
and success rate, and to provide additional properties to the selected aptamers. 26,27,28,29,30,31
Similar to protein antibody development, purified recombinant proteins or peptides expressed in
prokaryotic or eukaryotic systems can be used as targets for aptamers selected by the SELEX method.
However, because of the posttranslational modifications, especially in the case of highly glycosylated
proteins, purified proteins or peptides often cannot fold into the correct 3D structure that is formed
under physiologic conditions.32 Consequently, the newly synthesized aptamers may not be able to
selectively recognize and interact with their corresponding targets, which would result in failure of the
biomedical application. As this is a common problem, it is very important to choose biomarkers in their
native conformation for aptamers selection. Taking this issue into an account, a modified SELEX
technology that uses whole living cells, Cell-based SELEX (or Cell-SELEX), was recently
established.33 To develop cell-specific aptamers, the Cell-SELEX method uses whole living cells that
express surface biomarkers of interest. However, the presence of many different cell surface molecules
in addition to the target biomarker(s) results in the synthesis of many unrelated/unwanted aptamers.
Therefore, in addition to all the SELEX steps described above, Cell-SELEX technology also utilizes
control cells that do not express the target biomarker(s) during the counter-selection step. 33

Well-characterized biomarkers that are endogenously expressed at high levels, such as the ErbB
superfamily, MUC1, EpCAM, and CD30, offer the best potential for cell-based aptamer development.

Subsequently, cell lines that have high endogenous expression of cell-specific or cancer type-specific
biomarker(s) are commonly used for Cell-SELEX. However, if such cell lines are unavailable, a
biomarker of interest could be over-expressed in a particular cell line via gene transfection and the
parental cells used for counter-selection. Using this approach, aptamers targeting the cancer stem cell
(CSC) biomarker CD133 have been recently developed.34 In this study, CD133 cDNA was transfected
into HEK293T cells that were then used for aptamer enrichment, with the parental HEK293T cells
serving as a negative control. Similarly, an aptamer specific for the human receptor tyrosine kinase
was recently developed.35
Figure 2.
Schematic diagram of our hybrid-SELEX method for selection of CD30-specific ssDNA
aptamer. In our experiment, the hybrid-SELEX process is divided into (a) the cell-based SELEX
selection and (b) CD30 protein-based SELEX enrichment. First, CD30-expressing lymphoma cells are
used for positive selection and CD30-negative Jurkat cells are used in negative counter-selection. After
20 rounds of selection, the enriched aptamer pool is incubated with CD30 protein immobilized on
magnetic beads for five additional rounds of enrichment. SELEX, Systematic Evolution of Ligands by
EXponential enrichment.
Full figure and legend (183K)
Aptamers specific for cell surface biomarkers
Cell surface biomarkers are functionally important molecules involved in many biological processes,
such as signal transduction, cell adhesion and migration, cellcell interactions, and communication
between the intra- and extra-cellular environments. An abnormal expression of cell surface biomarkers
is often related to tumorigenesis.50 Clinically, it is estimated that about 60% of cancer-targeting drugs,
including therapeutic antibodies and small molecule inhibitors, target cell surface biomarkers, 51 making
them attractive for disease treatment. In the last decade, many aptamers targeting cell surface
biomarkers have been developed through the advancement of both the protein- and/or cell-based
SELEX technologies (see Table 2 for detailed list). These aptamers have been extensively studied for
diagnosis and/or treatment of hematological
malignancies,7,23,49 lung,52,53,54 liver,55 breast,56,57 ovarian,58 brain,59,60colorectal,61 and pancreatic
cancers,46 as well as for identification and characterization of CSCs. 34,62
Aptamer-Mediated Targeted Therapies
Traditional cancer treatment approaches, such as chemotherapy, radiotherapy, photodynamic therapy,
and photothermal therapy can cause serious side effects in patients due to their associated nonspecific
toxicity. To minimize these side effects, a concept of personalized, targeted therapy has been gaining

momentum. One of the main clinical approaches for targeted cancer therapy employs antibody-based
drugs. Although antibody-mediated therapy is highly specific and results in fewer side effects,
potential immunogenicity and high cost of production may limit its clinical applications. To overcome
these obstacles, oligonucleotide aptamer-based targeted therapeutics and specific drug delivery
systems have recently been explored. These studies revealed numerous advantages offered by the
aptamer technology over protein-based antibody therapies, with some of these described in the
section below.
Aptamer-drug conjugates
Aptamer-drug conjugation (ApDC) is a very simple yet effective model of noncovalently or covalently
conjugating aptamer sequences directly with therapeutic agents (Figure 3). For example, aptamerconjugated Doxorubicin (Dox), a chemotherapeutic agent extensively used in the treatment of various
cancers, has recently been shown to have enhanced therapeutic efficacy over Dox alone.
Mechanistically, Dox cytotoxicity is caused by its intercalation into the nucleic acid structure at the
preferred paired CG or GC sites with subsequent inhibition of cancer cell proliferation. Taking
advantage of its propensity for intercalation, Dox can be noncovalently conjugated to oligonucleotide
aptamers containing CG/GC sequences through a simple incubation step. A recent report by
Subramanian et al. describes the effectiveness of aptamer-Dox conjugates in the treatment of
retinoblastoma.63 In their study, a 2-fluoro modified RNA aptamer EpDT3 (specific for EpCAM, a CSC
marker), was noncovalently conjugated with Dox. After binding to EpCAM molecules expressed at the
cancer cell surface, the EpDT3-Dox conjugates were preferentially internalized by the cancer and not
by the healthy cells, greatly enhancing therapeutic efficacy and reducing treatment-associated side
effects. Several other studies also utilized aptamer-Dox conjugates for cancer therapy, such as HER2
aptamer-Dox conjugates targeting breast cancer,64 MUC1 aptamer-Dox conjugates targeting lung
cancer,65 and PSMA aptamer-Dox conjugates targeting prostate cancer.66 Despite their obvious
advantages, several concerns related to the use of aptamer-Dox conjugate have been raised. These
include (i) instability of the aptamer-drug conjugate due to the reversible nature of noncovalent
conjugation process; (ii) short circulating half-life of aptamer-drug conjugates in vivo due to their low
molecular weight; and (iii) poor drug payload capacity due to a very simple structure of aptamers.
These three disadvantages and technological approaches to improve them are described in greater
detail below.
Figure 3.
Schematic diagram of noncovalent or covalent aptamer-drug conjugation.
Full figure (40K)

To enhance the stability of drug loading, Dox can be covalently conjugated to aptamer sequences via a
functional linker moiety. For example, the DNA aptamer sgc8 possesses a strong affinity for PTK7
kinase that is abundantly expressed on the surface of CCRF-CEM T-cell acute lymphoblastic leukemia
cells. To enhance its stability, this aptamer was covalently conjugated with Dox through an acid-labile
linker.67 Once the sgc8 aptamer-Dox conjugate was preferentially bound and internalized by the target
cells, the acid-labile linker was easily cleaved in the acidic lysosomal environment, releasing Dox and
effectively killing target cells. 67 On the other side of the spectrum, covalent conjugation is the most
commonly used method of aptamer-drug conjugation, especially for agents that cannot intercalate into
the nucleic acid structure or whose intercalation would disrupt aptamer structure. 68 Evidence suggests
that these covalently conjugated aptamer-drug compounds are significantly more stable than the
corresponding noncovalently conjugated intercalations. 69
Conjugation of aptamers with high molecular weight polymers, such as polyethylene glycol (PEG), has
been examined in order to increase aptamer molecular weight. Specifically, PEG has been widely used
in drug modifications, including synthesis of Macugen aptamers. This modification, resulting in
PEGylated aptamers, not only increased the aptamer molecular weight and prolonged its circulating
half-life, but also enhanced its stability and decreased its toxic accumulation in nontarget tissues. 70,71
Finally, in order to increase aptamer-drug payload capacity, an innovative model named aptamertethered DNA nanotrains (aptNTrs) was recently introduced by Zhu et al. to deliver Dox to cancer
cells.72 In this study, structure of the sgc8 aptamer that targets PTK7 was modified by adding a DNA
trigger probe on the 5-end. Consequently, the modified aptamer acted as a locomotive for targeting,
while two hairpin monomers containing Dox intercalation sites acted as boxcars to deliver the drug.
After self-assembly, the newly synthesized sgc8 aptamer-NTrs displayed high drug payload capacity,
with the drug/sgc8 aptamer-NTr molar ratio of 50:1. Importantly, sgc8 aptamer-NTrs-Dox conjugates
were preferentially internalized by the target cells, thereby inhibiting tumor cell growth in vitro and in
vivo.72
Another strategy for increasing the aptamer payload capacity involves the construction of polyvalent
aptamers. Polyvalent aptamers exhibit an increased target affinity and are more rapidly internalized by
their target cells. To demonstrate this, Boyacioglu et al. developed a new DNA aptamer they termed
SZTI01 against PSMA.69 First, a dimeric aptamer complex (DAC) was created for specific delivery of Dox
to PSMA-expressing cancer cells. Then, the SZTI 01aptamer was modified on the 3-terminus with either
a dA16 or dT16 single-stranded tail that contained CpG sites for loading Dox, and the two monomers
were annealed in a 1:1 ratio to form the DAC structure. The results of the study showed that DACs
have a high Dox payload capacity with the Dox/DAC molar ratio of about 4:1, and the DACs-Dox

conjugates were stable under physiological conditions for up to 8 hours. 69 In another study, a DNA
aptamer targeting MUC1 was truncated and an aptamer containing three repeats of the active
targeting region, termed L3, was synthesized. Although the Dox payload capacity was not specifically
modified in the L3 aptamer, the L3-Dox conjugates showed a stronger affinity to target cells and lower
cytotoxicity to off-target cells than the parental MUC1 aptamer.73 Finally, polyvalent aptamers can also
be constructed through the rolling circle amplification (RCA) technology. Using the RCA method and
the sgc8 aptamer sequence as a circular template, a polyvalent sgc8 aptamer, termed Poly-AptamerDrug, was synthesized.74 It was determined that the Dox payload capacity of the polyvalent sgc8
aptamer increased tenfold, as compared to the monovalent sgc8 aptamer. Moreover, because of their
40-fold greater binding affinity, the Poly-Aptamer-Drug conjugates were more effective than their
monovalent counterparts in targeting and killing leukemia cells. 74
Although Dox presents itself as a very attractive chemotherapeutic agent for use in aptamer
conjugation, other drugs, such as Gemcitabine (Gem) and photosensitizers, can also be targeted to
cancer cells through the aptamer technology. Gem is an FDA-approved deoxycytidine analog (dFdC)
used for anticancer therapy. To deliver Gem specifically to pancreatic cancer cells, Ray et al. developed
a novel aptamer-Gem polymer model. In this model, a single-stranded RNA polymer contained Gem
that was enzymatically synthesized through a mutant T7 RNA polymerase-mediated transcription
reaction and fused with a nuclease-resistant 2-fluoro-modified RNA aptamer (E07) that selectively
binds to EGFR on pancreatic cancer cells. The E07 aptamer structure was modified by introducing a
24-nucleotide sequence at the 3 end and using it as an adaptor for Gem polymer binding. Following
an annealing step, the Gem polymer complementary bound with the E07 aptamer and preferentially
targeted the EGFR-expressing pancreatic cancer cells, inhibiting cell proliferation. 75
Compared with the traditional chemotherapeutic agents, controlled conditional prodrug
photosensitizers have also been extensively used for aptamer-mediated drug delivery. In this
therapeutic approach, termed photodynamic therapy, or photodynamic therapy, photosensitizers are
activated by light irradiation and induce production of intracellular reactive oxygen species, resulting in
cytotoxicity. A study by Ferreira et al. describes the development of a DNA aptamer specific for MUC1
and covalently conjugated at the 5 end with the photosensitizer chlorin e6. 76 Upon light irradiation,
MUC1-expressing epithelial cancer cells were preferentially killed with cytotoxicity about 500-fold
higher than that of the control cells. Similar studies have reported using a necleolin aptamer
(AS1411)-TMPyP4 for targeting breast cancer77 and the EGFR aptamer (R13)-TF70 for treatment of
lung cancer.78

Finally, approaches to extend the scope of aptamer application have also been developed. Similar to
bi-specific antibodies, bi-specific or even tri-specific aptamers can be constructed. A bi-specific
aptamer for targeting different cells was recently described by Zhu et al. In their study, specific DNA
aptamers sgc8 and sgd5a were conjugated through a dsDNA linker. Compared to each mono-aptamer,
this bi-specific aptamer (named SD) could recognize its target cell simultaneously with equal
specificity and affinity, while Dox intercalation into the dsDNA induced target cell cytotoxicity.79 In the
same study, a Y-shape dsDNA linker was used to construct a tri-specific aptamer that also recognized
its target cells with high specificity and affinity.79 Clinically, Min et al. proposed using a bi-specific
aptamer for prostate cancer therapy. It is well established that prostate tumors may contain both
PSMA-positive and -negative cell types. Thus, this study utilized two aptamers, a 2-fluoro modified
RNA aptamer targeting PSMA-expressing cells and a DUP-1 peptide aptamer specific to PSMA-negative
cells, conjugated through streptavidin. Moreover, intercalating Dox into the PSMA aptamer of this bispecific aptamer model could serve as a tool to target all prostate cancer cell types. 80
Aptamer-nanoparticle therapeutics
Nanoparticles (NPs) are attractive vehicles to increase both the half-life and the drug payload capacity
of aptamer-mediated drug delivery. In addition to their common features, such as biocompatibility for
clinical applications, large surface for enhanced aptamer and drug loading, and uniform size and shape
for excellent biodistribution, NPs have other individual physical and chemical properties defined by
their materials. For example, copolymers and liposomes are biodegradable, while metal materials offer
exceptional photothermal and magnetic performance.
Conclusion
Antibody-based targeted therapeutics provide high target specificity and affinity. However, their
potential for immunogenicity is of a great concern, as is their high production cost, both of which have
limited their clinical applicability. As discussed in this review, when compared to protein antibodies,
oligonucleotide aptamers offer many advantages, including simple chemical synthesis, virtual
nonimmunogenicity, smaller size, faster tissue penetration, ease of modification with different
functional moieties, low cost of production, and high biological stability. Therefore, aptamers have
become a promising new class of molecular ligands that could replace or supplement protein
antibodies. In summary, aptamer technology has a strong market value and may be applied in various
biomedical fields, including in vitro cancer cell detection, in vivo tumor imaging, and targeted cancer
therapy (Figure 7).
Figure 7.

Summary of various aptamer applications.

Full figure (58K)
Although aptamer technology has a great potential in the biomedical field, several technical challenges
remain and must be addressed. These include: (i) how can aptamers be rapidly adapted for specific
targets by decreasing false-positive/-negative selection? Primarily dependent on the natural properties
of targets of interest, such as proteins versus cells or tissues, the process of aptamer selection is
usually time-consuming, and the success rate is sometimes low. To improve the speed and success
rate, novel methods for aptamer selection have been recently described. They include bead-based
selection, that can select aptamers as rapidly as a single round of selection, 27,28 and the SOMAmer,
which improves the aptamer production success rate from less than 30% to over 50%. 29,30 More
recently, a study by Cho et al. devised a Quantitative Parallel Aptamer Selection System (QPASS)
method, which integrates microfluidic selection, NGS, and in situ-synthesized aptamer arrays. This
approach allows for the simultaneous measurement of affinity and specificity for thousands of
candidate aptamers in parallel.116 In addition to QPASS, evolving modifications to the Cell-SELEX
approach are beginning to address difficulties with successful removal of the influence stemming from
the presence of dead cells, slow enrichment aptamers recognizing targets of interest, and
contamination with unwanted aptamer sequences. As described above, utilization of the abovementioned FACS-mediated SELEX44,45 and hybrid-SELEX23 offers novel approaches that address these
technical challenges.
(ii) How can we select cancer-relevant targets for aptamer development and clinical applications?
Tumorigenesis is a dynamic process that includes multiple constantly changing factors. Therefore, a
one-size-fits-all cancer-specific biomarker is unlikely to ever be identified. Yet, it has been established
that certain biomarkers present in healthy tissues are highly expressed in cancer cells. Moreover,
certain biomarkers are associated with particular cancer cell types making them to be considered as
useful targets for development of targeted cancer therapy. However, while use of cancer cells to
identify biomarkers and to develop therapeutic agents is a reasonable approach, cultured cells,
especially immortalized cell lines, greatly differ from tumor tissues in vivo. To overcome these
limitations and to select more reliable cancer-relevant biomarkers for aptamer development, several
innovative SELEX methods have been recently described. Of particular interest are the tissue-based
SELEX117 and the in vivo-SELEX,118 which offer target selection under more relevant pathologic
conditions. This cell/tissue-specific biomarker selection can also be utilized for development of
noncancer related therapies, as shown for aptamers targeting the adipose tissue in obesity 119 and for
aptamers designed to penetrate the blood-brain barrier in order to combat brain diseases. 120 Hence,

we believe that the careful selection of cancer-associated biomarkers and cell/tissue type-specific
biomarkers will expand the scopes of aptamer applicability and improve the feasibility of clinical
applications.
(iii) What methods could improve aptamer biostability in vivo? Unmodified RNA-based aptamers are
very susceptible to the nuclease-mediated degradation in vivo. Although many chemical modifications
aimed at increasing biostability of the RNA aptamers have been developed, including 2-modifications,
3-modifications, phosphodiester backbone modifications, 19,20 and utilizations of novel nucleic acids
(locked nucleic acid and Spiegelmers),16,21,22 their effectiveness is still limited. When it was first
described, PEGylation was a very attractive strategy for prolonging aptamer circulation half-life and
enhancing their biostability. However, a recent report showed that the in vivo use of PEGylated
aptamers induced production of anti-PEG antibodies, 121 emphasizing the need for the development of
alternative approaches.
(iv) How can aptamer technology be modified to achieve a more effective drug delivery? Many drug
delivery systems described in this review are tested in vitroor in animal models. Yet, as with any
compound that is translated from the bench to the bedside, aptamer-drug conjugates may behave
differently in a human patient than they do in laboratory animals. Therefore, aptamer-drug
conjugation remains an important challenge that must be considered. Specifically, various coupling
approaches lead to different pharmacokinetics, biodistribution, and tolerability in vivo, which in turn
greatly affect treatment effectiveness. In the same vein, we must consider the effectiveness of
aptamer-mediated target gene therapy. Gene therapy, including siRNA and miRNA aimed at silencing
specific genes, is considered the next generation therapeutic approach. However, silencing a single
pathogenic gene may not be a viable therapeutic option because tumorigenesis is a process regulated
by multiple genes and signaling pathways. Therefore, combining targeted therapeutics with gene
therapy may represent the most effective strategy. Such combinational therapy approaches can
greatly improve the therapeutic efficacy while reducing the required dosages of both drugs and small
molecule RNAs,122 and, more importantly, may offer new alternatives to combat chemotherapyresistant cancers.110
(v) The last important point to consider is whether aptamer-mediated biotherapies can become
effective, FDA-approved medications. Following Macugen approval by the FDA, many aptamermediated biotherapies have been evaluated in clinical trials. Of particular interest is AS1411, an
antitumor aptamer that has completed several Phase I clinical trials. 15 Trial results are promising and
offer useful insights into further modifications that could be applied to therapeutic aptamer
development.

Taken together, although some technical challenges remain to be addressed, oligonucleotide aptamers
have become an attractive and promising tool for targeted cancer therapy. As more clinical data are
accumulated, we and others will be better equipped to optimize aptamer formulations, leading to the
expansion of aptamer use in the clinic.
9.1.5 Tumor Suppressors
Intrinsic Disorder in PTEN and its Interactome Confers Structural Plasticity and Functional
Versatility
Prerna Malaney, Ravi R Pathak, Bin Xue, VN Uversky & Vrushank Dav
Scientific Reports 20 June 2013; 3(2035)
http://dx.doi.org:/10.1038/srep02035
IDPs, while structurally poor, are functionally rich by virtue of their flexibility and modularity. However,
how mutations in IDPs elicit diseases, remain elusive. Herein, we have identified tumor suppressor
PTEN as an intrinsically disordered protein (IDP) and elucidated the molecular principles by which its
intrinsically disordered region (IDR) at the carboxyl-terminus (C-tail) executes its functions. Posttranslational modifications, conserved eukaryotic linear motifs and molecular recognition features
present in the C-tail IDR enhance PTENs protein-protein interactions that are required for its myriad
cellular functions. PTEN primary and secondary interactomes are also enriched in IDPs, most being
cancer related, revealing that PTEN functions emanate from and are nucleated by the C-tail IDR, which
form pliable network-hubs. Together, PTEN higher order functional networks operate via multiple IDPIDP interactions facilitated by its C-tail IDR. Targeting PTEN IDR and its interaction hubs emerges as a
new paradigm for treatment of PTEN related pathologies.
The concept of Intrinsic Disorder in proteins has rapidly gained attention as the preponderance and
functional roles of IDPs are increasingly being identified in eukaryotic proteomes 1, 2. Structured
proteins adopt energetically stable three-dimensional conformations with minimum free energy. In
contrast, IDPs, due to their unique amino acid sequence arrangements, cannot adopt energetically
favorable conformations and, thus, lack stable tertiary structure in vitro3. This structural plasticity
allows IDPs to operate within numerous functional pathways, conferring multiple regulatory
functions4, 5, 6. Indeed, mutations in and dysregulation of IDPs are associated with many diseases
including cancer1, 6, 7, signifying that IDPs play vital roles in functional pathways. Evidence suggests
that ~80% of proteins participating in processes driving cancer contain IDRs 6. For example, tumor
suppressor p53 as an IDP, functions via its C-terminal IDR, which simultaneously exists in different
conformations, each of which function differently 1. Since PTEN is the second most frequently mutated

tumor suppressor with versatile functions8, we hypothesized that PTEN may contain IDR(s) that can be
exploited for therapeutic targeting in cancers and diseases associated with pathogenic PI3K/Akt/mTOR
(Phosphoinositide 3-Kinase/Akt/ mammalian Target of Rapamycin) signaling 9, 10, 11.
PTEN (phosphatase and tensin homolog), a 403 amino acid dual protein/lipid phosphatase converts
phosphatidylinositol(3,4,5)-triphosphate (PIP3) to phosphatidylinositol(4,5)-bisphosphate (PIP2),
thereby regulating the PI3K/Akt/mTOR pathway involved in oncogenic signaling, cell proliferation,
survival and apoptosis12. PTEN, as a protein phosphatase, autodephosphorylates itself 13. Deficiency or
dysregulation of PTEN drives endometrial, prostate, brain and lung cancers, and causes neurological
defects14, 15. PTEN is activated after membrane association16, providing conformational accessibility to
the catalytic phosphatase domain (PD) that converts PIP3 to PIP2 16(Figure 1a). Because PTEN reduces
PIP3 levels and inhibits pathogenic PI3K signaling, therapeutically targeting PTEN to the membrane to
enhance its activity is of significance in treating several pathologies including cancer.
Figure 1: PTEN: A newly identified IDP.

PTEN A newly identified IDP. srep02035-f1

http://www.nature.com/srep/2013/130620/srep02035/images_article/srep02035-f1.jpg
(a) Diagrammatic representation of PTEN structure. PTEN, a 403 amino acid protein, comprises of
PBM: PIP2 Binding Module (AA 113; in green), a phosphatase Domain (AA 14185; in pink), C2
Domain (AA 190350; in blue), C-terminal region or Tail (AA 351400; in orange) and a PDZ binding
domain (AA 401403; in dark blue). The PDZ-binding motif is considered as a part of the C-terminal
region. *Figure not to scale. (b) Crystal structure of PTEN. Only the phosphatase (in pink) and C2

domain (in blue) are amenable to crystallization. The first seven residues and the last 50 residues
represent unstructured/loosely-folded regions that are yet to be crystallized. These regions represent
the N- and C-termini of PTEN, respectively. (Source: RCSB Protein Data Bank). (c) Disorder analysis of
PTEN. PONDR-VLXT and PONDR-FIT prediction tools were used to determine the disorder score of
PTEN. Any value above 0.5 indicates intrinsic disorder. There are several disordered stretches within
the PTEN protein, however, the most prominent of these disordered regions is a 50 amino-acid stretch
located at the C-terminus of the PTEN protein. (d) IDPs are enriched in polar (R, Q, S, T, E, K, D, H)
and structure breaking (G, P) amino acids and are depleted in hydrophobic (I, L, V, M, A), aromatic (Y,
W, F) and cysteine (C) and asparagine (N) residues. The amino acid sequence of PTEN highlights these
classes of residues with their relative distribution. (e) Composition profiling for full-length PTEN (in
green), its ordered domain (in yellow) and its IDR (in red). The tool used is Composition Profiler (Vacic
et al, 2007). As shown in the graph, the disordered region in PTEN is enriched in polar residues
(specifically H, T, D, S and E), structure breaking residues (specifically P) and is depleted in all
hydrophobic residues, cysteine and all aromatic residues. (f) Histogram representing the percentage of
hydrophobic, polar, aromatic, structure breaking, cysteine and asparagines residues in ordered vs.
disordered regions. The disordered region has an amino acid composition in line with the definition of
IDPs.

Full size image (751 KB)

PTEN crystal structure revealed that the PD and membrane-binding C2 domains are ordered (Figure
1b); however, the structures of the N-terminus, the CBR3 loop and the 50 amino-acid C-tail remain
undetermined17. The C-tail is of particular significance due to its ability to regulate PTEN membrane
association, activity, function, stability 18, 19, 20, 21. Herein, we identify PTEN as an IDP with its C-tail being
intrinsically disordered. The PTEN C-tail IDR is heavily phosphorylated by a number of kinases and
regulates the majority of PTEN functions, including a large number of PPIs that forms the PTEN
primary and secondary interactomes, comprising critical functional protein hubs, most of which are
related to cancer. Our analysis provides a mechanistic insight into the functioning of the PTEN C-tail
IDR at the systems level, including inter- and intra-molecular interactions that will aid in designing
drugs to enhance the lipid phosphatase activity of PTEN for the pharmacotherapy of cancers and
pathological conditions driven by hyperactive PI3K-signaling.
PTEN is an IDP
Utilizing two disorder prediction software programs, PONDR-VLXT and PONDR-FIT 22, 23, we have
identified PTEN as a bona fide IDP. PTEN has a highly disordered, functionally versatile, C-tail
encompassing amino acids 351403 (Figure 1a and 1c). A PDZ-binding motif (amino acids 401403) is

part of the disordered region. Thus, the PTEN C-tail IDR facilitates interactions with a vast repertoire
of PDZ domain-containing proteins (Figs. 1a and 2d). The unique amino acid composition of IDRs
dictates their structural plasticity3, 23, 24. IDRs are enriched in polar and structure-breaking amino acid
residues, depleted in hydrophobic and aromatic residues and, rarely, contain Cys and Asn
residues1, 23, 24. The ordered region of PTEN (AA 1350) has 25% hydrophobic, 43% polar, 9%
structure breaking, 13% aromatic and 9% Cys and Asn residues. In contrast, the PTEN C-tail (AA
351403) is enriched in polar (66%) and structure breaking (11%) residues and is depleted in
hydrophobic (11%), aromatic (6%) and Cys and Asn residues (6%), indicating an ideal profile for the
IDR (Figs. 1d and 1f ). Further, compositional analysis of PTEN using the Composition Profiler 24 reveals
that the disordered region in PTEN is enriched in polar residues (specifically H, T, D, S and E) and
structure breaking residues (specifically P) but is depleted in all aromatic and hydrophobic residues in
addition to cysteine. (Figure 1e), again exhibiting universal characteristics of IDPs. Taken together, we
establish the PTEN C-tail as a functional IDR and classify PTEN as a new IDP.
Figure 2: The functional relevance of the PTEN IDR.

The functional relevance of the PTEN IDR. srep02035-f2

http://www.nature.com/srep/2013/130620/srep02035/images_article/srep02035-f2.jpg
(a) The number of mutations observed in PTEN over its 403 amino-acid stretch is plotted. Fewer
mutations are observed in the tail region (in red) possibly indicating the deleterious nature of
mutations in the functionally critical C-terminal region. [Source: Sanger Institute Catalogue of Somatic
Mutations in Cancer (COSMIC), Human Gene Mutation Database (HGMD)]. (b) Number of mutations in
every successive 50 amino-acid stretch of the PTEN protein. The last 50 amino-acid stretch,

representing the tail region has at least one-eighth the number of mutations seen in any other 50
amino-acid stretch along PTEN, pointing to its critical function in cell homeostasis. (c) Correlation of
mutations with the amino acid composition of PTEN. The ratio of mutations in specific residues in the
disordered vs. ordered region are represented in this graph. The residues considered here are those
used to define IDRs: hydrophobic, polar, aromatic, structure-breaking, cysteine and asparagine
residues. Compared to the other classes of residues, mutations in aromatic residues are much higher
in the disordered region when compared to the ordered region. (d) The PTEN primary interactome.
Forty proteins interact with known regions of PTEN. There are approximately 340 more proteins that
interact with PTEN at sites that are yet to be determined (see Supplementary Table S2). Proteins
shown in pink interact with the phosphatase domain, those in blue interact with the C2 domain and
those in orange interact with the disordered tail. (Visualization tool: Cytoscape). (e) The PTEN C-tail
has a higher propensity for PPIs. Of the 40 mapped proteins, 60% interact with the disordered
indicating a strong correlation between degree of disorder and the number of protein interactions. (f)
Most proteins within the PTEN interactome are highly disordered. Approximately 80% of PTENinteracting proteins within the primary interactome are disordered, as indicated in red. The proteins
within the interactome that are ordered are indicated in blue.

Full size image (645 KB)

Low mutability of PTEN IDR suggests critical biological functions

Mutations in PTEN are associated with several types of cancers 14. To correlate PTEN mutations to its
structure, we analyzed all human PTEN mutations deposited in the COSMIC Database
(http://www.sanger.ac.uk/genetics/CGP/cosmic/). The disordered PTEN C-tail IDR shows unusually low
mutability (~8-fold less) compared to any other 50 amino-acid stretch of PTEN (Figure 2a and 2b). To
confirm our finding of the low mutability of the C-tail region, we also analyzed all human PTEN
mutations deposited in the Human Gene Mutation Database
(HGMD,http://www.hgmd.cf.ac.uk/ac/index.php)25 (Figure 2a), cBioPortal for Cancer
Genomics26, 27(Supplementary Figure S1) and the Roche Cancer Genome Database28 (Supplementary
Figure S1) which was consistent with the COSMIC database mutational data. It is likely that
evolutionary pressure maintains a survival advantage and ipso facto abrogates progeny with mutations
in highly functional protein sequences29, 30, 31. Thus, the functionally versatile PTEN C-tail IDR cannot
afford mutations, hence showing least number of mutations. It is equally likely that mutations in
individual residues within the IDR are well tolerated, as the evolutionary pressure may have shifted to
maintaining global biophysical properties and structural malleability of the IDR to safeguard the critical
protein function29. In either case, on a global scale, the versatile structural pliability of the PTEN IDR

dictates functional diversity and biological activities 29. Thus, the slightest functional perturbation in the
PTEN IDR due to mutations, either within the IDR or in domains interacting with it, could disrupt
cellular homeostasis as seen in cancers and neurodegenerative disorders associated with PTEN
mutations. This is supported by our data indicating that PTEN, as an IDP when mutated, causes
several cancers14.
Moreover, the PTEN C-tail IDR exhibits preferential mutations in aromatic residues compared to the
ordered region (Figure 2c). The ratio of mutations in aromatic residues in the disordered to ordered
region is much higher than any other class of residues (structure breaking, hydrophobic, polar, Cys
and Asn), likely attributed to the structure-imparting property of aromatic residue 32. Specifically,
aromatic residues within IDRs engage in stacking interactions, enhancing nucleation between distinct
residues at functional protein-protein interaction interfaces 32. Thus loss of this critical structural and
functional property imparted by aromatic residues is associated with a disease phenotype. In
summary, the disordered PTEN C-tail IDR has functionally evolved to contain a combination of
peptides that cannot tolerate mutations.
Disorderliness in PTEN primary interactome drives functional networks
Protein-Protein Interactions (PPIs) typically occur between conserved, structurally rigid regions of two
or more proteins, particularly ordered proteins that display energetically favorable, highly-folded
conformations. Intriguingly, IDPs lack tertiary structure, yet engage in PPIs, albeit with lower affinities
but high specificity1. The lack of structure within IDPs enhances their biophysical landscape, conferring
them with the ability to attain structural complementarities required for PPIs. Since IDPs do not
conform to a stable structure, they are less compact, providing a larger physical interface and
energetic adaptability to interact with multiple proteins1, 7. Thus, conditional folding within IDPs is
effectively utilized for interaction with a multitude of binding partners, enabling them to shuttle
between several signaling cascades as efficient cogs, mediating and regulating PPIs 4,7, 33, 34, 35, 36.
Indeed, we discovered that PTEN, being an IDP, interacted with more than 400 proteins
(Supplementary Table S1) when a combination of online software, literature search and database
mining tools were used. Proteins with known PTEN interaction domains were classified as mapped
(Figure 2d and Supplementary Table S1), whereas those with uncharacterized/predicted interactions
were designated as unmapped proteins (Supplementary Table S1). Derivation of PTEN primary
interactome from the mapped proteins using Cytoscape (http://www.cytoscape.org/) indicated that
PTEN disorderliness is efficiently used for interaction with 40 proteins, most existing in distinct
functional pathways (Figure 2d, 2e and Supplementary Table S2).

Interestingly, within the PTEN primary interactome, 60% of interactions occurred within the disordered
C-tail region. Furthermore, disorder analysis on the primary interactome revealed that 33 proteins
(>82%) were IDPs, of which two-thirds interacted with the C-tail IDR (Figure 2e,
2f andSupplementary Table S3), indicating a high propensity for disorder-disorder (D-D)-type
interactions.
In order to study evolutionary conservation of the PTEN C-tail and its interactions across species,
several sequence alignments were performed (Figure 3a). Sequence alignment of the entire PTEN
protein from different animal species shows a good conservation of the catalytic phosphatase domain
between vertebrates and invertebrates with 100% sequence conservation for the dual specificity
phosphatase catalytic motif HCKAGKGR8 (Supplementary Figure S2). The C-tail shows good
conservation in the vertebrate species, likely indicating the recent emergence of the function of PTEN
C-tail region in regulating PTEN activity and enriching its PPI potential, translating to its versatile
functions. In order to examine the conservation across species for the PTEN C-tail interacting proteins,
a literature search was conducted to identify experimentally verified domains/motifs involved in
interaction with the C-tail. The domains involved in these interactions with the C-tail for 13 proteins
with relevant literature sources for these interactions are part ofSupplementary Figure S3. Subsequent
sequence alignments for these thirteen proteins (Supplementary Figure S3) shows good sequence
homology for the domains/motifs involved in interaction with the PTEN C-tail. These findings support
the concept that the PTEN C-tail has evolved in vertebrates to incorporate features that allow it to
interact with these proteins.
Figure 3: Sequence conservation in PTEN and its interacting partners reflects functionality.

Sequence conservation in PTEN and its interacting partners reflects functionality. srep02035-f3

http://www.nature.com/srep/2013/130620/srep02035/images_article/srep02035-f3.jpg
(a) Sequence alignment of the PTEN protein for vertebrate and invertebrate animals. Green color
indicates sequence similarity while red indicates sequence dissimilar amino acid residues. All
comparisons are made with respect to the human PTEN protein. (b) Network analysis for PTEN was
performed to assess its potential as a network hub. The network shows multiple secondary
interactions within the 40 mapped proteins, indicating their role in multiple signaling cascades

mediated via PTEN. The proteins SMAD2/3, AR, PCAF, ANAPC7, B-arrestin 1 and p53 appear to be
critical within these signaling cascades and also happen to be intrinsically disordered (Supplementary
Table S3), reinforcing the concept of preferential interactions between disordered proteins. (Analysis
Tool: Metacore by GeneGo).

Full size image (819 KB)

Further, to assess whether PTEN acts as a functional hub protein and regulates pathways through its
protein-binding partners, we performed functional network analysis using the Analyze Network option
from MetaCore (GeneGo Inc, Thomson Reuters, 2011) (Figure 3b). The PTEN primary interactome was
used as input with PTEN as the central node. We identified multiple interactions not only between
PTEN (node) and SMAD2/3, AR, PCAF, ANAPC3, ANAPC4, Caveolin, -arrestin 1 and p53 (edges), but
also amongst the edge proteins themselves (Figure 3b). Interestingly, all the edge proteins are
themselves highly disordered (Supplementary Table S3). Further supporting this finding, our functional
enrichment revealed that 13 proteins (one-third) of the PTEN primary interactome were cancer-related
and highly disordered (Figure 4a, Supplementary Table S3 and S4).
Figure 4: Derivation and disorder analysis of the PTEN cancer interactome.

Derivation and disorder analysis of the PTEN cancer interactome. srep02035-f4

http://www.nature.com/srep/2013/130620/srep02035/images_article/srep02035-f4.jpg

Derivation of the PTEN Cancer Interactome. Functional enrichment of the PTEN

primary interactome identified 13 cancer-related proteins which are also intrinsically
disordered. Subsequently, the PTEN secondary interactome was derived from the
primary PTEN interacting proteins. A subset of the secondary interactome was
designated as the PTEN Cancer Interactome and it represents the proteins that interact

with the 13 cancer-related proteins of the primary interactome. (b) PTEN Cancer
Interactome. PTEN is the primary node that interacts with the 13 cancer-related
proteins representing the partial primary interactome. Proteins that interact with each
of the 13 cancer-related proteins comprise the secondary interactome. Disordered
proteins are represented in red while ordered proteins are shown in blue. Cancerrelated proteins in the PTEN primary interactome were identified using IPA
(IngenuitySystems, ingenuity.com). (c) We identified 40 proteins that are part of the
PTEN primary interactome of which 13 are highly disordered (IDP) and identified as
potential cancer network hubs based on functional network analysis. We further
identify 299 IDPS from the secondary PTEN interactome. A filter for cancer-related
proteins revealed that approximately two-thirds of the IDPs that form the secondary
interactome (193 out of 299) are involved in oncogenesis, suggesting a high degree of
functional enrichment. (Functional network analysis was performed using IPA
(Ingenuity Systems,www.ingenuity.com).Full size image (805 KB)
Pliant PTEN secondary interactome relays function of the primary network
The disorderliness of the PTEN primary interactome prompted us to investigate the possibility that
PTEN radiates its function via a malleable network of IDPs that extends beyond the primary
interactome. Therefore, we derived the PTEN secondary interactome (Supplementary Table S5) and
ascertained the interaction of 13 cancer-related proteins identified in the primary interactome (Figure
4a). The entire PTEN secondary interactome consisted of 299 IDPs, of which 193 IDPs (two-thirds)
were associated with the 13 cancer-related proteins, generating a PTEN-Cancer Interactome (Figure
4, Supplementary Table S5 and S6). Thus, two-third of the IDPs within the PTEN secondary
interactome associates with one-third of the cancer related IDPs within the PTEN primary interactome,
indicating that cancer-related functions are driven by IDPs in the PTEN interactome and that the
flexibility of IDP-IDP interactions modulates diverse functions; dysregulation of which causes cancers.
Functional network analysis of the 193 cancer-related IDPs identified 31 proteins that shared multiple
nodes (Figure 5a and Supplementary Table S6). We overlaid this network with the cancer-related IDPs
of the primary interactome to predict functionally critical protein hubs (indicated in yellow circles
in Figure 5a and b). Our analysis revealed 16 proteins as highly populated hubs, most enriched in
disordered regions, again demonstrating that a high degree of structural and functional association
between the hubs required IDP-IDP interactions (Figure 5b). The involvement of these hubs in
multiple, critical oncogenic signaling pathways make them attractive drug targets in the field of clinical
oncology. Our bioinformatic analysis resonates well with observed biological phenomena as seen in the
case of MDM2 protein, which is a major PPI hub regulating p53. Interaction of the human androgen
receptor (AR) protein and MDM2 influences prostate cell growth and apoptosis 37. Mdm2-Daxx
interaction activates p53 following DNA damage38, and Daxx binds and inhibits AR function39.
Conversely, the breast cancer susceptibility gene 1 (BRCA1) interacts directly with AR and enhances

AR target genes, such as p21(WAF1/CIP1), that may result in the increase of androgen-induced cell
death in prostate cancer cells40. Further, BRCA1 complexes with Smad3 and is inactivated, leading to
early-onset familial breast and ovarian cancer41. Within the same network, MDM2 inhibits the
transcriptional activity of SMAD proteins including SMAD3 42, thereby, emerging as a major player in
prostrate, breast and ovarian cancer. Loss of PTEN, on the other hand, results in resistance to
apoptosis by activating the MDM2-mediated antiapoptotic mechanism. We also identified proteins like
NCL, DAXX and SUMO that play critical roles in mediating cancers as being a part of the PTEN centric
cancer interactome (Figure 5b). Interestingly, all of the 16 predicted hubs can be traced back to PTEN
(either directly or through other signaling adaptors) reinforcing our analysis (Figure 5c). These
findings support the prevailing concept of preferential interaction between disordered regions of two
distinct proteins; with PTEN being the common disordered interacting hub, giving functional centrality
to PTEN in many critical cellular pathways.
Figure 5: Predicting functionally relevant network hubs in the PTEN cancer interactome.

Predicting functionally relevant network hubs in the PTEN cancer interactome. srep02035-f5

http://www.nature.com/srep/2013/130620/srep02035/images/srep02035-f5.jpg
(a) Methodology to identify functional hubs within the PTEN Cancer Interactome. The PTEN Cancer
Interactome contains 193 IDPs that are potential hubs. Over-represented IDPs (or IDPs with multiple
occurrences) in the PTEN Cancer Interactome would have a greater propensity to function as hubs.
Upon sorting for over-represented IDPs the list of 193 proteins is brought down to 31 proteins. In
order to assess the possibility of these 31 proteins as functional hubs a network analysis is warranted.

(b) We identified 31 potential hubs based on multiple associations from within the 193 cancerassociated IDPs of the PTEN secondary interactome. Regulatory networks derived from these 31
proteins were overlaid with a similar network from the 13 cancer-related proteins. Based on the
number of associations within the network, we identify 16 potential functional hubs in the PTEN cancer
interactome (indicated in yellow). Regulatory interactions were generated using the Transcriptome
Browser tool (Lopez et al, 2008). (c) Functional network analysis of the 16 predicted hubs. In order to
assess the functional association of the 16 predicted hubs with PTEN a network analysis with PTEN
as a central node was done. The analysis identifies MDM2 protein, a major regulator of p53, as one of
the major PPI hubs in the PTEN cancer interactome. A number of other critical cancer-related proteins,
such as AR, SMAD2/3 and PDGFRB that are part of the PTEN primary interactome, feature prominently
in the PTEN cancer interactome. We also identified proteins like NCL, DAXX and SUMO that play critical
roles in mediating cancers as being a part of the PTEN centric cancer interactome. Interestingly, all of
the 16 predicted hubs can be traced back to PTEN (either directly or through other signaling adaptors)
reinforcing our analysis. (Functional network analysis was performed using IPA
(IngenuitySystems, www.ingenuity.com).

Full size image (799 KB)

To further validate our methodology in using intrinsic disorder and cancer as filters to identify key
signaling hubs, we compared our data sets with a previously published cancer signaling data set. We
derived 7 common hubs (Supplementary Table S7), which were extended using the expansive human
signaling network described previously43, 44, 45, 46 to obtain the PTEN associated cancer interactome
(Figure 6a). An extensive disease associated network analysis using IPA validated our predictions as
all the seven predicted hubs had an extensive cross-talk across multiple cancer disease types (Figure
6b).
Figure 7: Biochemical features modulating PTEN PPIs.

Biochemical features modulating PTEN PPIs. srep02035-f7

http://www.nature.com/srep/2013/130620/srep02035/images_article/srep02035-f6.jpg
(a) A PTEN linked cancer network was derived using seven of the 16 predicted cancer hubs that were
common with the human cancer associated gene set. The associated partners of the seven hubs were
extracted from the human signaling network (Cui et al, 2007, Awan et al, 2007, Li et al, 2012 and
Newman et al, 2013). Red color denotes the potential cancer hubs and blue color are their associated
partners. Topological analysis identifies p53 as the most significant network hub in the PTEN linked
cancer network (Supplementary Table S7). (b) Disease associated network of PTEN cancer hubs. A

functional network was constructed with the seven topologically relevant hubs identified previously
using the Core Analysis function from the IPA suite to derive the primary network (denoted as MP). A
disease network was constructed using the Path Designer option and disease associated biological
functions were overlaid on the primary network. Fx denotes the different functions associated with the
members of the networks.

Full size image (923 KB)

Modulation of PTEN PPIs by linear binding motifs

Recent evidence has shown that IDPs mediate PPIs via short linear amino acid sequences (~20
residues) called Molecular Recognition Elements (MoREs) or Molecular Recognition Features
(MoRFs)35, 47. MoRFs undergo disorder-to-order transitions upon binding and adopt thermodynamically
stable well-defined structures47, increasing the propensity of IDPs to interact with a vast repertoire of
proteins. MoRFs also display molecular recognition elements that capture the binding partner proteins
with high specificity. These partner-dependent conformational differences are critical to imparting
versatile binding properties to IDRs35.
Since the PTEN IDR engages in multiple PPIs, we tested the possibility for the existence of MoRFs. The
MORFP red algorithm48 revealed that PTEN contains major MoRF sites at amino acids 273279 (part of
the disordered CBR3 loop of the C2 domain), amino acids 339347 (in close vicinity of the disordered
C-tail) and amino acids 395403 (part of the disordered C-tail) (Figure 7a and Supplementary Figure
S4). The primary restriction of MoRFs to the PTEN C-tail IDR or adjacent regions indicates that these
MoRFs directly participate in modulating PPI functions (Figure 7a). However, mutational analysis within
MoRFs is required to establish their active role in functional PPIs.
Figure 7: Biochemical features modulating PTEN PPIs.

Biochemical features modulating PTEN PPIs. srep02035-f7

http://www.nature.com/srep/2013/130620/srep02035/images_article/srep02035-f7.jpg
(a) MoRFs in the PTEN C-tail IDR. MoRFpred (Disfani et al, 2012), a computational tool, was used to
identify MoRF regions within the PTEN protein (Supplementary Figure S4). The MoRFs in the vicinity of
the C-tail IDR are highlighted in red. Interestingly, all of the major MoRFs (with a length greater than
5 residues) are observed in the vicinity of disordered regions (either part of the disordered CBR3 loop
of the C2 domain or the C-tail IDR) indicating a positive correlation between intrinsic disorder and
PPIs. (b) ELMs in PTEN C-tail IDR. Eukaryotic Linear Motifs (or ELMs) are 311 amino acid long

sequences that mediate PPIs. IDRs are particularly enriched in ELMs (Dinkel et al, 2012). The linear
motifs occurring in the disordered segment of PTEN (tail + PDZ domain) have been highlighted. The
motifs with a high conservation score (>0.75) are indicated in red. Interestingly, all of the motifs with
a high conservation score are restricted to the C-tail IDR. (c) Phosphorylation sites in the C-tail IDR.
Phosphorylation of PTEN, particularly on serine and threonine residues in the disordered region,
regulates the function and stability of PTEN. Phosphorylation occurs at Ser 362, Thr 366, Ser 370, Ser
380, Thr 382, Thr 383, Ser 385 by various enzymes such as Casein Kinase II, Glycogen synthase
kinase 3-B and Polo-like kinase 3. Each of these phosphorylation events helps regulate the availability
and stability of the PTEN molecule within the cell.

Full size image (342 KB)

Protein-protein interactions are also facilitated by very short motifs (310 amino acids) called Short
Linear Motifs (SLiMs) or Eukaryotic Linear Motifs (ELMs) 49, 50. Because of their short sequences, ELMs
arise/disappear by simple point mutations, providing the evolutionary plasticity that the ordered
protein domains lack. Thus, ELMs easily adapt to novel interactions in signaling pathways, where rapid
assembly/disassembly of multi-protein complexes is a prerequisite. The frequent occurrence of ELMs
in a typical proteome indicates their critical cellular functions. Consistent with this notion, a higher
density of ELMs are observed in hub proteins and IDPs 50. Since ELMs have short sequences, they
interact with low-affinity, however, they engage in highly cooperative binding in protein complexes,
triggering productive signaling50. Therefore, at increased intracellular local concentrations they
competitively bind to mutually overlapping physiological targets of each other as seen with PDZ, SH2
and PTB interaction domains found in cancer-associated proteins and in IDRs 49, 50. As PTEN contains a
PDZ-binding motif within the IDR (Figure 1a and c), we probed for the existence and features of ELMs
in PTEN using The Eukaryotic Linear Motif Resource (http://elm.eu.org). We identified 34 different
classes of ELMs in PTEN that mediate PPIs (Supplementary Figure S5). Interestingly, the four ELMs
that are most conserved (conservation score>0.75) occurred within the PTEN C-tail IDR, indicating its
high level of functional/biological significance (Figure 7b). ELM functions are further modulated by
post-translational modifications, mainly by phosphorylation50. Indeed, the PTEN IDR possesses nine
phosphorylation sites51, 52(Figure 7c).
PTEN phosphorylation modulates intramolecular association and PPI function
Post-translational Modifications (PTMs) in IDPs facilitate PPIs 5. Modifying enzymes readily dock on
structurally flexible IDRs, making them a hot spot for PTMs 4, 7, 53, 54. Consistent with this notion,
regulatory cancer-associated proteins have twice as much disorder and undergo more frequent
phosphorylation/dephosphorylation than other cellular proteins as predicted by DISPHOS (a DISorder-

enhanced PHOSphorylation prediction software)54, implicating a tight interconnection between protein

phosphorylation and disorder. Consistent with the function of PTM in IDRs, clustering of Ser and Thr
phosphorylation sites (Figure 7c) in the C-tail IDR regulates PTEN stability, membrane association and
activity19, 20. Phosphorylation in the PEST [proline (P), glutamic acid (E), serine (S) and threonine (T)]
domain within the C-tail IDR (amino acids 352 to 399) inhibits degradation of PTEN 51. Casein kinase II
(CK II), Glycogen synthase kinase 3-beta (GSK3-) and PLK3 (Polo-like kinase 3) phosphorylate Ser
and Thr residues within the IDR, each providing a distinct function 51 (Figure 7c). The microtubuleassociated serine/threonine (MAST), serine/threonine kinase 11(STK11) or LKB1 and casein kinase I
(CKI) kinases have also been implicated in PTEN phosphorylation. STK11/LKB1 modifies T383, while
CKI modifies T366, S370 and S38552. Indeed, our DISPHOS prediction for C-tail IDRs supports these
experimental observations (Supplementary Figure S6).
Substrate-kinase interactions are typically of the disordered-ordered (D-O) type and are stabilized by
hydrogen bonding (Figure 7c), a hallmark of IDRs54. Indeed, computational analysis revealed that
large ordered regions comprising the catalytic domains of CKII, GSK3B, PLK3, Rak, and Src kinases
interact with the C-tail IDR (Supplementary Table S8), indicating that PTEN engages in D-O type
intermolecular interactions with the modifying kinases.
At the intramolecular level, phosphorylation at C-tail residues triggers a conformational change in
PTEN, inhibiting its membrane association and, therefore, its lipid phosphatase activity 18, 19, 21, 55. The
phosphorylated C-tail IDR folds onto the PD and C2 domains giving rise to the closed-closed
conformation of PTEN (Figure 8a) that is incapable of interaction with the membrane 18, 20. The closedclosed form of PTEN is enzymatically inactive and cannot convert PIP3 to PIP2. The identification of
the exact resides involved in this intramolecular interaction remains an active area of research 18, 20, 56.
Figure 8: Targeting PTEN C-tail IDR.
http://www.nature.com/srep/2013/130620/srep02035/images_article/srep02035-f8.jpg
Most PTEN functions emanate from the C-tail IDR, including aberrant PPIs that hyper-activate
oncogenic pathways. (a) Phosphorylation mediates an intramolecular interaction in the PTEN molecule.
Phosphorylation causes a conformational change in PTEN converting it to the enzymatically inactive
closed closed form wherein the flexible tail folds onto residues in the C2 and phosphatase domain,
thereby making it incapable of interacting with the membrane. Dephosphorylation (by an unknown
phosphatase or via auto-dephosphorylation) converts PTEN to the open-closed form. Electrostatic
interactions, mediated by the PBM, further convert PTEN to the open-open form wherein it binds to
the membrane and acts as a lipid phosphatase converting PIP3 to PIP2, thereby, abrogating signaling
via the PI3K/Akt/mTOR pathways. Subsequent to membrane binding, several E3 ubiquitin ligases

polyubiquitinate PTEN marking it for proteasomal degradation. Phosphorylation, by inducing the

intramolecular interaction, masks the ubiquitination sites thereby increasing the half-life of the PTEN
protein within the cell. Therefore, phosphorylation negatively regulates PTEN function but positively
regulates its stability. (b) PTEN IDR engages in PPIs of the disorder:order type (D-O type). As revealed
in the present study, this occurs via the use of a MoRF or SLiM region. Therefore, designing a
peptidomimetic drug molecule that competes with the PTEN MoRF/SLiM binding to the ordered protein
will abrogate PTEN binding, therefore PTEN function. PTEN IDR is highly accessible to multiple kinases
that phosphorylate and modulate PTEN function, mainly its inhibition via intra-molecular interactions.
PTEN inhibition hyper-activates the PI3K/AKT/mTOR pathway, which increase the oncogenic potential
of the cell and drives cancer growth. Therefore, targeting the PTEN C-tail IDR with small molecules
that bind and sterically hinder PTEN phosphorylation and/or intra-molecular interactions will be an
ideally adjunctive therapy to multiple inhibitor therapy targeting of the PI3/AKT/mTOR pathway.

Full size image (553 KB)

It was recently shown that the phosphorylation events of PTEN occur in two independent cascades of
ordered events, with the S380S385 cluster being modified prior to the S361S70 cluster 52. Even
within the two clusters, the phosphorylation events follow a specific pattern with a distributive kinetic
mechanism. Not surprisingly, distributive kinetics is energetically favorable on protein domains that
are highly disordered with multiple ensembles of flexible structures 52. Thus the dynamic nature of
these phosphorylation events is contingent to the inherent flexibility in the PTEN structure driven by
intrinsically disordered C-tail crucial for PTEN stability and localization within the cell (Figure 8a).
Targeting intrinsic disorder in PTEN and its interactome
Drug targeting to critical protein regions can mitigate aberrant cellular processes driving
oncogenesis57. However, despite numerous clinical trials with molecularly targeted therapies, failure
rates for cancer treatments remain high. Conventional therapies targeting pathway-specific kinases
suffer from off-target effects and often fail due to the emergence of compensatory and alternative
pathways58. As a novel approach, facile drug targeting to IDRs within critical signaling hub proteins is
highly plausible59, 60, 61. Moreover, as IDRs undergo extensive PTMs53 and engage in PPIs4, 34, 36, the
multitude of resulting protein interactions (normal and aberrant) can be targeted concomitantly with a
cocktail of distinct inhibitors, which dampens oncogenic signaling 60.
Indeed, targeting PPIs is a more selective treatment strategy over conventional enzyme inhibitors 60.
However, disruption of multiple ordered interfaces within PPIs by small molecule inhibitors remains
challenging62. The advantage of targeting IDPs engaged in PPIs is that, unlike ordered proteins, they
engage in PPIs via MoRFs or ELMs, which are small peptide regions that bind with low affinity and thus

are susceptible to disruption by small molecule inhibitors 59. Consistent with this notion, small
molecules disrupted highly disordered complexes of p53-Mdm2 and c-Myc-Max interactions by
inducing order upon binding60, 63. Likewise, targeting the PTEN C-tail IDR may reduce its intra- and
inter-molecular interactions and limit accessibility to enzymes mediating PTMs (Figure 8b), providing a
means to increase PTEN activity. Our analysis shows that since the C-tail IDR is rich in conserved
MoRFs/SLiMS, targeting these regions will prove to be a rational therapeutic modality for a large
number of cancers that show compromised PTEN activity or hyperactivation of the oncogenic
PI3K/AKT/mTOR pathway9, 10, 11. Since reductions in the levels and activity of PTEN are sufficient to
drive oncogenesis11, 14, 15, increasing PTEN activity is an ideal therapy for cancers associated with
hyperactive PI3K-signaling.

Discussion
Recent studies on genome- and proteome-wide molecular alterations in diseases indicate that
pathological conditions are caused by perturbations in complex, highly interconnected biological
networks64. Thus, current reductionist approach of studying structure-function relationship in diseases
has limited our abilities to discover effective targeted therapeutics. In an attempt to overcome these
limitations, in the current study, we have undertaken a novel approach to drug discovery that exploits
systems and network biology at the structural, topological and functional level. Using PTEN, a tumor
suppressor, we have applied computational and systems biology approaches and integrated extensive
data-mining and biochemical properties of IDP interactions to reach a finer understanding of PTEN
function. These results have identified PTEN C-tail IDR and several hub proteins in PTEN-driven
molecular network implicated in human diseases as therapeutic targets, enhancing the repertoire of
clinically relevant biological targets for pharmacotherapy.
Our derivation and analysis of PTEN primary and secondary interactome indicates that altered levels or
interactions of IDPs perturb myriad cellular signaling pathways, leading to pathological conditions
including cancer. IDPs have the propensity to aggregate and cause cellular toxicity 65. Therefore, PTEN
as an IDP has evolved a mechanism, wherein, the level of active PTEN, its cellular localization and
PTEN-PPIs are regulated via phosphorylation of the C-tail IDR. Furthermore, evolutionarily conserved
ELMs and MoRFs that we have identified within the C-tail IDR may play a critical role in orchestrating
the formation and function of the PTEN interactome.
Increase in complexity of PPIs is either directed by the number and type of proteins or by increasing
the number of interactions required to execute cellular functions 66. To delineate how PTEN executes
myriad functions, we first derived the PTEN primary interactome. We found 40 proteins to directly
interact on the PTEN molecule, out of which 25 were associated with the C-tail IDR, consistent with

the concept that disorderliness within PTEN executes its myriad functions. To enhance our
understanding of PTEN functions in the context of multiple distinct pathways at the systems-level, we
delineated functional networks operating within the primary interactome. Our findings showed a high
degree of cross-talk between edges, implying that shared regulatory modules, comprised of multiple
signaling cascades, operate via PTEN-mediated interaction networks. When these networks are
altered, diseases ensue with extreme functional penalties. We also found that the edge proteins were
themselves highly disordered indicating that disorderliness within the PTEN primary interactome
confers functional versatility. Supporting this notion, 13 proteins that were functionally classified as
cancer-related were also highly disordered forming a pliable PTEN-Cancer Interactome. Thus, PTEN
lesions influence the flexibility of IDP-IDP interactions modulating diverse functions, likely causing
cancer.
Owing to the inherent ability of PPIs to be flexible while being complex, specific cellular functions are
readily fine-tuned as per the biological demands. Emerging evidence suggests that certain features on
the IDRs are recognized as a way of conferring plasticity to protein interaction networks. Consistent
with this concept, our data suggest that PTEN, a hub protein containing an IDR, likely utilizes MoRFs
and ELMs, gets differentially modified via PTMs, acquiring complementary structures to engage and
modulate PPI activity by facilitating adaptive binding to multiple protein partners in many cellular
pathways. Thus, our present work provide a novel entre in targeting intrinsic disorder in PTEN and its
interactome to dampen the aberrant PI3K-signaling that drives many cancers. First, imparting order to
the PTEN structure may help dampen multiple oncogenic signaling pathways mediated via the 16 hub
proteins identified in the present study, by limiting their affinity for PPIs. Second, targeting intrinsic
disorder in PTEN and its interactome can become an adjunctive or alternative approach to the use of
various kinase inhibitors, which are toxic and have many off-target effects when used to mitigate the
aberrant hyperactivation of PI3K/AKT/mTOR oncogenic signaling pathway. Taken together, the present
findings provide a novel entre to design strategies for drug discovery and may become a logical
intervention in the pharmacotherapy of cancer and other PTEN-associated disease treatment
modalities.
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Notes from Opening Plenary Session The Genome and Beyond from the
2015 AACR Meeting in Philadelphia PA; Sunday April 19, 2015
April 30, 2015 by sjwilliamspa

Notes from Opening Plenary Session The Genome and Beyond from the 2015
AACR Meeting in Philadelphia PA; Sunday April 19, 2015

Reporter: Stephen J. Williams, Ph.D.

The following contain notes from the Sunday April 19, 2015 AACR Meeting (Pennsylvania Convention
Center, Philadelphia PA) 9:30 AM Opening Plenary Session

The Genome and Beyond

Session Chairperson: Lewis C. Cantley, Ph.D.
Speakers: Michael R. Stratton, Tyler Jacks, Stephen B. Baylin, Robert D. Schreiber,Williams R. Sellers
1.

A) Insights From Cancer Genomes: Michael R. Stratton, Ph.D.; Director of the

Wellcome Trust Sanger Institute

How do we correlate mutations with causative factors of carcinogenesis and exposure?

Cancer was thought as a disease of somatic mutations

UV skin exposure see C>T transversion in TP53 while tobacco exposure and lung
cancer see more C>A transversion; Is it possible to determine EXPOSURE
SIGNATURES?

Use a method called non negative matrix factorization (like face pattern recognition but
a mutation pattern recognition)

Performed sequence analysis producing 12,000 mutation catalogs with 8,000 somatic
mutation signatures

Found more mutations than expected; some mutation signatures found in all cancers,
while some signatures in half of cancers, and some signatures not found in cancer

For example found 3 mutation signatures in ovarian cancer but 13 for breast cancers
(80,000 mutations); his signatures are actually spectrums of mutations

kataegis: defined as localized hypermutation; an example is a signature he found

related to AID/APOBEC family (involved in IgG variability); kataegis is more prone in
hematologic cancers than solid cancers

recurrent tumors show a difference in mutation signatures than primary tumor before
drug treatment

1.

B) Engineering Cancer Genomes: Tyler Jacks, Ph.D.; Director, Koch Institute for
Integrative Cancer Research

Cancer GEMs (genetically engineered mouse models of cancer) had moved from
transgenics to defined oncogenes

Observation that p53 -/- mice develop spontaneous tumors (lymphomas)

then GEMs moved to Cre/Lox systems to generate mice with deletions however these
tumor models require lots of animals, much time to create, expensive to keep;

figured can use CRSPR/Cas9 as rapid, inexpensive way to generate engineered mice
and tumor models

he used CRSPR/Cas9 vectors targeting PTEN to introduce PTEN mutationsin-vivo to

hepatocytes; when they also introduced p53 mutations produced hemangiosarcomas;
took ONLY THREE months to produce detectable tumors

also produced liver tumors by using CRSPR/Cas9 to introduce gain of function mutation
in -catenin

See an article describing this study by MIT News A New Way To Model Cancer: New gene-editing
technique allows scientists to more rapidly study the role of mutations in tumor development.
The original research article can be found in the August 6, 2014 issue of Nature[1]
And see also on the Jacks Lab site under Research
1.

C) Above the Genome; The Epigenome and its Biology:Stephen B. Baylin

Baylin feels epigenetic therapy could be used for cancer cell reprogramming

Interplay between Histone (Movers) and epigenetic marks (Writers, Readers) important
for developing epigenetic therapy

Difference between stem cells and cancer: cancer keeps multiple methylation marks
whereas stem cells either keep one on or turn off marks in lineage

Corepressor drugs are a new exciting class in chemotherapeutic development

(Histone Demythylase {LSD1} inhibitors in clinical trials)

Bromodomain (Brd4) enhancers in clinical trials

1.

D) Using Genomes to Personalize Immunotherapy: Robert D. Schreiber, Ph.D.,

The three Es of cancer immunoediting: Elimination, Equilibrium, andEscape

First evidence for immunoediting came from mice that were immunocompetent
resistant to 3 methylcholanthrene (3mca)-induced tumorigenesis but RAG2 -/- form
3mca-induced tumors

RAG2-/- unedited (retain immunogenicity); tumors rejected by wild type mice

Edited tumors (arent immunogenic) led to tolerization of tumors

Can use genomic studies to identify mutant proteins which could be cancer specific
immunoepitopes

MHC (major histocompatibility complex) tetramers: can develop vaccines against

epitope and personalize therapy but only good as checkpoint block (anti-PD1 and anti
CTLA4) but personalized vaccines can increase therapeutic window so dont need to
start PD1 therapy right away

For more details see references Schreiker 2011 Science and Shankaran 2001 in Nature

1.

E) Report on the Melanoma Keynote 006 Trial comparing pembrolizumab

and ipilimumab (PD1 inhibitors)

Results of this trial were published the morning of the meeting in the New England Journal of Medicine
and can be found here.
A few notes:
From the paper: The antiPD-1 antibody pembrolizumab prolonged progression-free survival and
overall survival and had less high-grade toxicity than did ipilimumab in patients with advanced
melanoma. (Funded by Merck Sharp & Dohme; KEYNOTE-006 ClinicalTrials.gov
number, NCT01866319.)
And from Twitter:
Robert Cade, PharmD @VTOncologyPharm
KEYNOTE-006 was presented at this weeks #AACR15 conference. Pembrolizumab blew away
ipilimumab as 1st-line therapy for metastatic melanoma.
2:02 PM 21 Apr 2015
Jeb Keiper @JebKeiper
KEYNOTE-006 at #AACR15 has pembro HR 0.63 in OS over ipi. Issue is ipi is dosed only 4 times over
2 years (per label) vs Q2W for pembro. Hmm
11:55 AM 19 Apr 2015
OncLive.com @OncLive
Dr Antoni Ribas presenting data from KEYNOTE-006 at #AACR15 Read more about the findings,
at http://ow.ly/LMG6T
11:25 AM 19 Apr 2015
Joe @GantosJ
$MRK on 03/24 KEYNOTE-006 vs Yervoy Ph3 stopped early for meeting goals of PFS, OS & full data
@ #AACR15 now back to weekend & family
9:05 AM 19 Apr 2015
Kristen Slangerup @medwritekristen
Keytruda OS benefit over Yervoy in frontline #melanoma $MRK stops Ph3 early & data to come
@ #AACR15 #immunotherapy http://yhoo.it/1EYwwq8
2:40 PM 26 Mar 2015
Yahoo Finance @YahooFinance

Mercks Pivotal KEYNOTE-006 Study in First-Line Treatment for

Merck , known as MSD outside the United States and Canada, today announced that the randomized,
pivotal Phase 3 study investigating KEYTRUDA compared to ipilimumab in the first-line treatment
of
View on web

Stephen J Williams @StephenJWillia2

Progression free survival better for pembrolzumab over ipilimumab by 2.5
months#AACR15 @Pharma_BI #Cancer #Immunotherapy
11:56 AM 19 Apr 2015

Stephen J Williams @StephenJWillia2

Melanoma Keynote 006 trial PD1 inhibitor #Immunotherapy 80% responders after 1
year @Pharma_BI #AACR15

References
1.

Xue W, Chen S, Yin H, Tammela T, Papagiannakopoulos T, Joshi NS, Cai W, Yang G,

Bronson R, Crowley DG et al: CRISPR-mediated direct mutation of cancer
genes in the mouse liver. Nature2014, 514(7522):380-384.

Other related articles on Cancer Genomics and Social Media Coverage were published in this
Open Access Online Scientific Journal, include the following:

Cancer Biology and Genomics for Disease Diagnosis

Introduction The Evolution of Cancer Therapy and Cancer Research:
How We Got Here?
Methodology for Conference Coverage using Social Media: 2014 MassBio
Annual Meeting 4/3 4/4 2014, Royal Sonesta Hotel, Cambridge, MA
List of Breakthroughs in Cancer Research and Oncology Drug Development
by Awardees of The Israel Cancer Research Fund
2013 American Cancer Research Association Award for Outstanding
Achievement in Chemistry in Cancer Research: Professor
Alexander Levitzki
Genomics and Epigenetics: Genetic Errors and Methodologies Cancer
and Other Diseases

Cancer Genomics Leading the Way by Cancer Genomics Program at UC

Santa Cruz
Genomics and Metabolomics Advances in Cancer
Pancreatic Cancer: Genetics, Genomics and Immunotherapy
Multiple Lung Cancer Genomic Projects Suggest New Targets, Research
Directions for Non-Small Cell Lung Cancer

Posted in Cancer and Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy,Clinical
Genomics, Conference Coverage with Social Media, CRISPR/Cas9 & Gene Editing,Health Economics and Outcomes
Research | Tagged AACR, breast cancer, cancer, Cancer immunotherapy, cancer models, Clinical
Trials, GEMM, gene mutations, genetics, genomics,Harvard Medical School, medicine, Melanoma, mouse
model, National Institutes of Health,PD1, Personalized medicine, research, science, Stem cell | Leave a comment

Notes On Tumor Heterogeneity: Targets and Mechanisms, from the 2015

AACR Meeting in Philadelphia PA
April 29, 2015 by sjwilliamspa

Notes On Tumor Heterogeneity: Targets and Mechanisms, from the 2015 AACR
Meeting in Philadelphia PA
Reporter: Stephen J. Williams, Ph.D.
The following contain notes from the Sunday April 19, 2015 AACR Meeting (Pennsylvania Convention
Center, Philadelphia PA) 1 PM Major Symposium Session on Tumor Heterogeneity: Targets and
Mechanism chaired by Dr. Charles Swanton.
Speakers included: Mark J. Smyth, Charles Swanton, Ren H. Medema, andCatherine J. Wu
Tumor heterogeneity is a common feature of many malignancies, especially the solid tumors and can
drive the evolution and adaptation of the growing tumor, complicating therapy and resulting in
therapeutic failure, including resistance. This session at AACR described the mechanisms, both genetic
and epigenetic, which precipitate intratumor heterogeneity and how mutational processes and
chromosomal instability may impact the tumor progression and the origin of driver events during
tumor evolution. Finally the session examined possible therapeutic strategies to take advantage of,
and overcome, tumor evolution. The session was chaired by Dr. Charles Swanton. For a more complete
description of his work, tumor heterogeneity, and an interview on this site please click on the link
below:

Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed
by Multiregion Sequencing
and
Issues in Personalized Medicine: Discussions of Intratumor Heterogeneity from the Oncology Pharma
forum on LinkedIn

Notes from Charles Swanton, Cancer Research UK; Identifying Drivers of Cancer
Diversity
Dr. Swantons lecture focused on data from two recent papers from his lab by Franseco Favero and
Nicholas McGranahan:
1.

Glioblastoma adaptation Traced Through Decline of an IDH1 clonal driver and

macro-evolution of a double-minute chromosome(Annals of Oncology, 2015)[1]

This paper described the longitudinal Whole Genome Sequencing (WGS) study of a 35 year old female
whose primary glioblastoma (GBM) was followed throughtemozolomide treatment and ultimately
recurrence.

In 2008 patient was diagnosed with primary GBM (three biopsies of unrelated sites
were Grade II and Grade IV; temozolomide therapy for three years then relapse in
2011

WGS of 2 areas of primary tumor showed extensive mutational and copy number
heterogeneity; was able to identify clonal TP53 mutations and clonal IDH1 mutation in
primary tumor with different patterns of clonality based on grade

Amplifications on chromosome 4 and 12 (PDGFRA, KIT, CDK4)

After three years of temozolomide multiple translocations found in chromosome 4 and

12 (6 translocations)

Clonal IDH1 R132H mutation in primary tumor only at very low frequency in recurrent
tumor

The WGS on recurrent tumor (sequencing took ONLY 9 days from tumor resection to
sequence results) showed mutation cluster in KIT/PDGFRA.PI3K.mTOR axis so patient
treated with imatinib

However despite rapid sequencing and a personalized approach based on

WGS results, tumor progressed and patient died shortly:tumor evolution is
HUGE hurdle for personalized medicine

As Dr. Swanton stated:

we are underestimating the frequency of polyclonal evolution

1.

Clonal status of actionable driver events and the timing of mutational processes in
cancer evolution (Science Translational Medicine, 2015)[2]

analyzed nine cancer types to determine the subclonal frequencies of driver events, to
time mutational processes during cancer evolution, and to identify drivers of subclonal
expansions.

identified later subclonal actionable mutations,

including BRAF (V600E),IDH1 (R132H), PIK3CA (E545K), EGFR (L858R),
and KRAS (G12D), which may compromise the efficacy of targeted therapy
approaches.

> 20% of IDH1 mutations in glioblastomas, and 15% of mutations in genes in the PI3K
(phosphatidylinositol 3-kinase)AKTmTOR (mammalian target of rapamycin) signaling
axis across all tumor types were subclonal

Mutations in the RASMEK (mitogen-activated protein kinase kinase) signaling axis

were less likely to be subclonal than mutations in genes associated with PI3K-AKTmTOR signaling

Branched chain can converge on single resistance mechanism; clonal resistance (for example to PI3K
inhibitors can get multiple PTEN mutations in various metastases
Targeting Tumor Heterogeneity

Identify high risk occupants (have to know case history)

Mutational landscape interferes with anti-PD1 therapies

Low frequency mutations affect outcome

Notes from Dr. Catherine J. Wu, Dana-Farber Cancer Institute: The evolutionary
landscape of CLL: Therapeutic implications

Clonal evolution a key feature of cancer progression and relapse

Hypothesis: evolutionary dynamics (heterogeneity) in chronic lymphocytic leukemia

(CLL) contributes to variations in response and disease tempo

Used whole exome sequencing and copy number data of 149 CLL cases to discover
early and late cancer drivers: clonal patterns (Landau et. al, Cell 2013); some drivers
correspond to poor clinical outcome

Methylation studies suggest that there is epigenetic heterogeneity which may drive CLL
clonal evolution

Developing methodology to integrate WES to determine mutations with immunogenic

potential for development of personalized immunotherapy for CLL and other
malignancies

References
1.

Favero F, McGranahan N, Salm M, Birkbak NJ, Sanborn JZ, Benz SC, Becq J, Peden
JF, Kingsbury Z, Grocok RJ et al: Glioblastoma adaptation traced through
decline of an IDH1 clonal driver and macro-evolution of a double-minute
chromosome.Annals of oncology : official journal of the European Society for
Medical Oncology / ESMO 2015, 26(5):880-887.

2.

McGranahan N, Favero F, de Bruin EC, Birkbak NJ, Szallasi Z, Swanton C: Clonal

status of actionable driver events and the timing of mutational processes in
cancer evolution. Science translational medicine 2015, 7(283):283ra254.

Other related articles on Tumor Heterogeneity were published in this Open Access
Online Scientific Journal, include the following:

Issues in Personalized Medicine: Discussions of Intratumor Heterogeneity

from the Oncology Pharma forum on LinkedIn
Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and
Branched Evolution Revealed by Multiregion Sequencing
CANCER COMPLEXITY: Heterogeneity in Tumor Progression and Drug
Response 2015 Annual Symposium @Koch Institute for Integrative
Cancer Research at MIT W34, 6/12/2015 9:00 AM EDT 4:30 PM EDT
My Cancer Genome from Vanderbilt University: Matching Tumor
Mutations to Therapies & Clinical Trials
Tumor Imaging and Targeting: Predicting Tumor Response to Treatment:
Where we stand?
Mitochondrial Isocitrate Dehydrogenase and Variants
War on Cancer Needs to Refocus to Stay Ahead of Disease Says
Cancer Expert
Posted in Biological Networks, Biological Networks, Gene Regulation and Evolution, Cancer and
Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Informatics,Cancer Screening, Cell
Biology, Cell Biology, Signaling & Cell Circuits, Clinical Genomics,Computational Biology/Systems and
Bioinformatics, Conference Coverage with Social Media, Gene Regulation, Genome Biology, Immuno-Oncology &
Genomics, Scientific & Biotech Conferences: Press Coverage, Translational Research | Tagged AACR, breast
cancer, cancer, cancer genomics, Cancer immunotherapy, clonal heterogeneity, driver
mutations, genomics, Gleevec, Glioblastoma, Harvard Medical School, health, IDH1 and IDH2, Personalized
medicine, research, science, Temozolomide, tumor heterogeneity,Whole genome sequencing | Leave a comment

Upcoming Meetings on Cancer Immunogenetics

August 22, 2014 by sjwilliamspa

Upcoming Meetings on Cancer

Immunogenetics
Curator: Stephen J. Williams, Ph.D.

Below is a curation of upcoming 2014-15 Cancer Immunogenetics symposia. Some listed have CME
credits.

August 2014
Target Discovery for T Cell Therapy Symposium
Next Step to Advance Immunotherapies
August 14, 2014 | Part of ImVacS The Immunotherapies and Vaccine Summit
Learn more | View Agenda PDF | Register by July 18 & SAVE up to $200
Q&A with Dr. Adrian Bot of Kite Pharma

SITC 2014 Meetings

The Society for Immunotherapy of Cancer (SITC) is a 501 (c)(3) non-profit society of medical
professionals. Recent advances in immunology and biology have opened up new horizons in the field
of cancer therapy, with an upsurge in the integration of new biologic agents into clinical practice. With
several high-caliber scientific meetings with a focus on clinical and translational aspects of biologic
approaches to cancer treatment and numerous networking opportunities unique to this organization,
the Society for Immunotherapy of Cancer (SITC) has developed into the premier destination for
interaction and innovation in the cancer biologics community.

Upcoming SITC Meetings and Activities

Advances in Cancer Immunotherapy (ACI) Regional CME-Certified Programs

La Jolla, CA Friday, August 22, 2014

Portland, OR Friday, October 3, 2014
Charlotte, NC Friday, October 3, 2014
Tampa, FL Friday, December 5, 2014

September 2014

Hematologic Malignancies: Translating Discoveries to Novel Therapies

September 20-23, 2014 Sheraton Philadelphia Downtown Philadelphia, PA
The AACR is proud to announce our conference focused on the blood-based cancers and associated disorders

categorized as hematologic malignancies. Sessions will include presentations on leukemia, lymphoma, myeloma
myelodysplastic syndrome, and myeloproliferative neoplasms.

Advances in Melanoma: From Biology to Therapy

Loews Philadelphia Philadelphia, PA September 20-23, 2014
With so many recent advances in treating metastatic melanoma, including approaches like immunotherapies,
targeted therapies, and combination therapies, melanoma research is at a critical point where it is extremely
important for the field to have a continuous exchange of information. Despite the success of various targeted
inhibitors, therapeutic responses in melanoma patients are often short-lived due to rapidly acquired drug
resistance. Therefore, it is essential that melanoma researchers translate the novel understanding of melanoma
biology to decipher the mechanisms of innate and acquired drug resistance for the development of improved
therapeutic options. To bridge the gap between scientists and clinician-scientists professional practice, this
conference will provide a platform for discussion and potential collaborations for the discovery of new
therapeutic targets.

The 4th Mastering Immunogenicity Summit

September 15-16, 2014

British Consulate-General, Boston MA, USA
Join leaders in the immunogenicity field for a two day conference to learn what constitutes a successful strategy
for managing immunogenicity risk, and explore the business case for introducing immunogenicity assessment
into your program.

Learn about the latest strategies and exciting new technologies

Discuss current and developing challenges and exchange new ideas
Improve the outcome of your R&D programs

Our 4th Mastering Immunogenicity Conference will continue to have a strong focus on immunogenicity
sciences, particularly on what basic research needs to be carried out to improve our understanding of immune

regulation to biotherapeutics. We will review progress made in correlating data from pre-clinical predictive tools
to clinical outcomes, as well as continuing our discussions surrounding the benefits that Quality by Design has
on reduced immunogenicity, considering subsequent patient benefits as well as competitive advantage.
Presentations by experts will provide an overview of the wide range of technologies currently used for
immunogenicity risk management and how they can be incorporated for a quality by design approach.

Immunogenomics 2014
September 29 October 1, 2014
HudsonAlpha Biotechnology Campus
Huntsville, Alabama, USA
The HudsonAlpha-Science Conference on Immunogenomics will bring together preeminent leaders and
thinkers at the intersection of genomics and immunology.

October 2014

Cancer Immunotherapy: Out of the Gate

October 06, 2014 Grand Hyatt New York Hotel at Grand Central, New York, NY

The Cancer Research Institute (CRI) will host its 22nd Annual International Cancer Immunotherapy
Symposium October 6-8, 2014 at The Grand Hyatt in New York City. Attracting clinicians, laboratory
scientists, postdoctoral fellows, and graduate students, the symposium will feature plenary
presentations from leaders in immunology and cancer immunotherapy, a poster session, and
numerous networking opportunities.
This years CRI symposium, entitled Cancer Immunotherapy: Out of the Gate, will harness the
excitement and enthusiasm generated by recent clinical successes to explore new and emerging areas
of basic, translational, and clinical research. Topics such as the use of genomic methods to catalogue
cancer heterogeneity, mechanistic studies of checkpoint blockage antibodies, new views on
immunosurveillance and immunoregulation, and emerging therapies that are altering the landscape of
cancer treatment will be discussed.
See more at: http://www.cancerresearch.org/grants-programs/conferences-meetings/annualinternational-cancer-immunotherapy-symposia/2014-symposium#sthash.PnY56e5E.dpuf
Cytokines 2014
October 2629, Melbourne, Australia
EMBO Conference: Innate Lymphoid Cells
September 29October 1, Paris, France
Recommended reading
Laurie Dempsey

November 2014
SITC 2014 November 6-9, 2014

Gaylord National Hotel & Convention Center, National Harbor, MD

SITC 29th Annual Meeting
SITC Workshop on Combination Immunotherapy: Where Do We Go From Here?
SITC Primer on Tumor Immunology and Cancer Immunotherapy
SITC Hot Topic Symposium including two topics explored concurrently:
o Accelerating Tumor Immunity with Agonist Antibodies
o Engineered T Cell Toxicities
Professional Development Session: A Roadmap for Thriving in Your Career

The Fourth International Conference on Regulatory T cells and TH Subsets and Clinical Application in
Human Diseases

November 14, Shanghai, China

Recommended reading
Olive Leavy

Keystone Symposium: Cell Death Signaling in Cancer and the Immune System
October 28-November 2, Sao Paolo, Brazil
Recommended reading

December 2014
Tumor Immunology and Immunotherapy: A New Chapter
Co-Chairpersons: Robert H. Vonderheide, Nina Bhardwaj, Stanley Riddell, and Cynthia L. Sears
December 1-4, 2014 Orlando, FL

2015 Conferences
Keystone Symposia on Molecular and Cellular Biology
Tumor Immunology: Multidisciplinary Science Driving Combination Therapy
February 813, 2015
Fairmont Banff Springs, Banff, Alberta, Canada

March 2015
1.

813, Montreal, Quebec, Canada

2.

2227, Banff, Alberta, Canada

3.

293 April, Snowbird, Utah, USA

9th World Immune Regulation Meeting

Keystone Symposium: The Golden Anniversary of B Cell Discovery
Recommended reading
Keystone Symposium: T Cells: Regulation and Effector Function
Recommended reading

Posted in Academic Publishing, BioSimilars, CANCER BIOLOGY & Innovations in Cancer Therapy, Health Economics
and Outcomes Research, Human Immune System in Health and in Disease, Immunodiagnostics, Innovation in
immunology diagnostics, Monoclonal Immunotherapy, Scientific & Biotech Conferences: Press Coverage |
Tagged AACR, Cancer immunology, Cancer immunotherapy, drug discovery, emerging
technologies, genetics,health, immunology, immunotherapy, medicine, Personalized medicine, research, Scientific
& Biotech Conferences: Press Coverage | Leave a comment

CANCER BIOTHERAPEUTICS @ Proteins & Antibody Engineering

Summit, 4-8, 11, 2013, Lisbon, Portugal
June 13, 2013 by 2012pharmaceutical

Reporter: Aviva Lev-Ari, PhD, RN

CANCER BIOTHERAPEUTICS
ADCs, Multi-Specifics, Combined Therapies and Immunotherapy
Inaugural
TUESDAY , 5 NOVE MBER
PRE-CONFERENCE PLENARY SESION
16:55 Designing Receptor Binding Proteins with Highly Potent
Biological Function
Andreas Plckthun, Ph.D., Director and Professor, Biochemistry, University
of Zurich
Non-IgG molecules, unless armed with toxins or other effector units, are
usually thought to be limited in the biological responses they can elicit.
However, Designed Ankyrin Repeat Proteins (DARPins) are particularly
versatile, because of their favorable biophysical properties, and they can be
engineered into many formats. Using DARPins generated against members
of the EGFR family, and a combination of x-ray crystallography, signaling

studies, and in vivo experiments, it will be demonstrated how molecules

could be engineered to selectively induce apoptosis in tumors, and their
mechanism of action has been deduced. New intracellular sensors will be
described for such studies.
17:45 Immunotherapy with BiTE Antibodies
Luis Borges, Ph.D., Scientific Director, Therapeutic Innovation Unit, Amgen, Inc.
BiTE antibodies are potent bispecific single-chain antibodies that redirect T
cells to kill tumors. They engage a tumor target and a constant region of the
T cell receptor to recruit and activate polyclonal T cells to eliminate tumors.
They have demonstrated potent efficacy in various preclinical tumor models
and have now transitioned to clinical studies. Blinatumomab, a CD19xCD3
BiTE antibody, is in clinical development and has shown high single-agent
response rates in patients with refractory or relapsed B-ALL and B-NHL.
18:30 End of Day
Wednesday, 6 November
07:45 Registration and Morning Coffee
08:30 Chairpersons Opening Remarks
Jason Baum, Ph.D., Principal Scientist, Research, Merrimack Pharmaceuticals, Inc.
MULTI-SPECIFIC ANTIBODY PRODUCTS
08:35 Two-in-One Antibody Targeting EGFR and HER3 and Platform
Update
Germaine Fuh, Ph.D., Senior Scientist, Antibody Engineering, Genentech, Inc.
Mutation at the antigen binding sites of a mono-specific antibody may recruit a
second binding specificity such that each Fab arm exhibits dual binding function and
IgG with this dual action Fab (DAF) can be produced as conventional IgG. Proofof-

concept is a HER2/VEGF Two-in-One antibody; EGFR/HER3 Two-in-One DAF

antibody is in clinical phase II trial for treating epithelial cancer. The talk will cover the
generation and development of the EGFR/HER3 DAF antibody including preclinical
and clinical phase I data.
09:05 MM-141, a Bispecific Antibody Co-Targeting IGF-1R and Erbb3,
Overcomes Network Adaptation by Blocking Redundant Survival
Pathways
Jason Baum, Ph.D., Principal Scientist, Research, Merrimack Pharmaceuticals, Inc.
An integrated Network Biology approach was used to design and optimize MM141 to overcome limitations of first generation IGF-1R therapies by also blocking
heregulin-mediated compensation through ErbB3. MM-141 potentiates the activity
of both targeted therapies and chemotherapies through the combined inhibition
of PI3K/Akt/mTOR signaling as well as control over feedback loops triggered by
these agents.
09:35 Bispecific -bodies for Selective Inhibition of CD47 in Cancer Cells
Nicolas Fischer, Ph.D., Head, Research, Novimmune SA
We have used our -body platform to generate CD47-neutralizing bispecific
antibodies. These fully human antibodies are composed of a CD47-specific arm
and a targeting arm, specific to a tumor associated antigen (TAA). The preferential
neutralization of CD47 on TAA-expressing cancer cells should therefore show better
pharmacological properties and a broader therapeutic window as compared to nontargeted
anti-CD47 monoclonal antibodies. The presentation will also highlight how
light chain diversity can be exploited to create bispecific antibodies with favorable
manufacturability and stability profiles that facilitate their development path.
10:05 Sponsored Presentation (Opportunity Available)

10:35 Coffee Break in the Exhibit Hall with Poster Viewing

11:05 Targeting Tumor Microenvironmental Signals with Bispecific
Antibodies
Alessandro Angelini, Ph.D., David H. Koch Institute for Integrative Cancer Research,
Massachusetts Institute of Technology (MIT)
We have developed bispecific antibodies that locally contravene soluble signaling
factors that establish the supporting tumor microenvironment that enables tumor
survival and growth. Soluble factors such as VEGF, TGF-, and IL-8 play a demonstrated
role in tumorigenesis, and enhanced interdiction of these signals within the tumor
should enhance the therapeutic index of cancer therapy.
11:35 Novel Multi-Targeting Antibody Mixtures: Mode of Action and
Advantages Over Other Approaches
Michael Kragh, Ph.D., Director, Antibody Pharmacology, Symphogen A/S
This talk will present the selection of antibodies against tumor-related antigens to
obtain synergistic combinations, the benefits of simultaneous targeting of multiple
receptors, and examine pan-HER (EGFR, HER2 and HER3) targeting to address
tumor heterogeneity and plasticity.
12:05 Sponsored Presentation (Opportunity Available)
12:35 Luncheon Presentations (Sponsorship Opportunities Available) or
Lunch on Your Own
ADVANCES WITH CANCER IMMUNOTHERAPY
14:00 Chairpersons Remarks
Andrea van Elsas, CSO, BioNovion B.V.
14:05 Cancer Immunotherapy Using Immune Modulating Antibodies
Andrea van Elsas, CSO, BioNovion B.V.
Immune rejection of human cancer has been an elusive goal until recently. T cell
modulating antibodies targeting CTLA-4 and the PD-1 pathway induced clinically
meaningful responses and long-term benefit in patients with metastatic cancer.

Successful immune rejection can come with significant immune related adverse
events. Immune oncology agents do not directly tumor cells but treat the patients
immune cells. In this presentation, the discovery of immune modulating antibodies
and their translation into clinical success will be discussed.
14:35 Immunocytokines: A Novel Potent Class of Armed Antibody
Laura Gualandi, Ph.D., Philochem A.G.
Antibodies are effective tools that can deliver molecules with potent therapeutic
activity, such as Cytokines, to the tumor site, minimizing toxic effects. Aspects like
molecular format, valence and the chosen target antigen contribute to the efficacy of
the immunocytokines in vivo. Combinatory therapeutic strategies with other agents
have also been recently investigated. This talk will cover advanced preclinical and
clinical data on armed antibodies discovered and developed by the Philogen group.
15:05 NKTT320: A Humanized Monoclonal Antibody for Cancer
Immunotherapy
Robert Mashal, CEO, NKT Therapeutics
Activation of iNKT cells has been shown to have therapeutic effects both in
PEGSummitEurope.com 7
6-7 November 2013
preclinical models and in patients with cancer, and represents an important pathway
for the immunotherapy of cancer. iNKT cells have an invariant T cell receptor (iTCR).
NKT Therapeutics is developing NKTT320, a humanized monoclonal antibody which
specifically recognizes the iTCR present exclusively on iNKT cells, and has been
shown to activate iNKT cells both in vitro and in vivo.
15:35 Refreshment Break in the Exhibit Hall with Poster Viewing
16:15 Novel Tumor-Targeted, Engineered IL-2 Variant (IL-2v)-Based

Immunocytokines for Immunotherapy of Cancer

Ekkehard Moessner, Ph.D., Group Leader, Protein Engineering, pRED, Roche Glycart A.G.
A novel class of immunocytokines will be discussed that are based on Fc containing
and also on non-Fc containing building blocks. The IL2 component is optimized for
improved performance in tumor targeting. Enhancement of in vivo efficacy, when
combined with ADCC competent antibodies, will be discussed.
ANTIBODY-DRUG CONJUGATES AND PAYLOADS
16:45 Next-Generation ADCs: Enabling Higher Drug Loading,
Alternative Payloads, and Alternative Targeting Moieties
Timothy B. Lowinger, Ph.D., CSO, Mersana Therapeutics, Inc.
The application of polymers to antibody-drug conjugate (ADC) design can provide
numerous advantages, including significantly higher capacity for drug payload;
utilization of alternative payloads not suitable for direct conjugation; improvement of
physicochemical properties; and utilization of protein recognition scaffolds beyond
the commonly used IgGs. Examples of these benefits achieved using Mersanas
polyacetal-based conjugation system to create next-generation ADCs
will be presented.
17:15 Problem Solving Roundtable Discussions
Table 1: Engineering of Bispecific Antibodies
Moderator: Nicolas Fischer, Ph.D., Head, Research, Novimmune SA
Table 2: Antibody-Drug Conjugates: Linkers and Payloads
Moderators: Robert Lutz, Ph.D., Vice President, Translational Research &
Development, ImmunoGen, Inc.
Timothy B. Lowinger, Ph.D., CSO, Mersana Therapeutics, Inc.
Table 3: Site-Specific Conjugation of ADCs
Moderator: Pavel Strop, Ph.D., Associate Research Fellow, Protein
Engineering, Rinat-Pfizer, Inc.
Table 4: Cancer Immunotherapy: Reaping the Benefits

Moderators: Andrea van Elsas, CSO, BioNovion B.V

Luis Borges, Ph.D., Scientific Director, Amgen, Inc.
Table 5: Cancer Biotherapeutics in the Clinic
Moderators: Jason Baum, Ph.D., Principal Scientist, Research, Merrimack
Pharmaceuticals, Inc.
Martine Piccart, M.D., Ph.D., Head, Medical Oncology, Jules Bordet
Institute; Chair, ESMO (European Society for Medical Oncology)
18:15 Networking Reception in the Exhibit Hall with Poster Viewing
19:15 End of Day One
Thursday, 7 November
07:45 Breakfast Presentation (Sponsorship Opportunity Available) or
Morning Coffee
08:30 Chairpersons Remarks
Robert Lutz, Ph.D., Vice President, Translational Research & Development,
ImmunoGen, Inc.
08:35 A Universal Chemically Driven Approach for Constructing
Homogeneous ADCs
David Jackson, Ph.D., Principle Scientist, ADC Discovery, Igenica, Inc.
Current ADCs in clinical development are heterogeneous mixtures that differ in
both DAR (drugs/antibody) and their conjugation sites. Igenica has invented novel
site-specific linkers to enable the synthesis of homogeneous ADCs. The linkers
are compatible with a variety of drug payloads and can be applied to any antibody.
Homogeneous ADCs were synthesized using the novel linkers and compared to
heterogeneous ADCs made with conventional linkers. Analytical data and activity of
the ADCs in tumor models will be presented.
09:05 Location Matters: Site of Conjugation Modulates Stability and
Pharmacokinetics of Antibody-Drug Conjugates
Pavel Strop, Ph.D., Associate Research Fellow, Protein Engineering, Rinat- Pfizer, Inc.

To understand the role of conjugation site, we developed an enzymatic method for

site-specific antibody-drug conjugation. This allowed us to attach diverse compounds
at multiple positions and investigate how the site influences stability, toxicity, and
efficacy. We show that the conjugation site has significant impact on ADC stability
and pharmacokinetics in a species-dependent manner. With this method, it is
possible to produce homogeneous ADCs and tune their properties to maximize the
therapeutic window.
09:35 Development of Second Generation Duocarmycin ADCs with
Superior Therapeutic Window
Marion Blomenrhr, Ph.D., Program Manager Biopharmaceuticals, Synthon
Biopharmaceuticals
The first generation ADCs have successfully exploited the mAb-driven tumor cell
targeting for optimization of efficacy, but have failed to reduce off-target toxicities.
This presentation will highlight Synthons second generation Linker-Drug technology
and its complementarity with novel proprietary duocarmycin payloads yielding highly
stable and potent ADCs, with an improved in vivo therapeutic window.
10:05 Producing Better Antibody-Drug Conjugates Sponsored by
(ADCs) Using ThioBridge Conjugation
Antony Godwin, Ph.D., Director, Science & Technology, PolyTherics Ltd
Next-generation antibody-drug conjugates will be required to be less heterogeneous
and have better stability. PolyTherics has developed ThioBridge for improved
conjugation of a cytotoxic payload at the disulfides bonds of antibodies, antibody
fragments and other targeting proteins. With ThioBridge, the resulting ADC
has the benefit of reduced heterogeneity, as the drug to antibody ratio is limited
to a maximum of 4 with little DAR 0 species. Stability is also enhanced, as unlike

single thiol conjugation approaches at disulfides, ThioBridge is not prone to

drug deconjugation reactions in serum. In vitro and in vivo data for mAb and Fab
conjugates with an established payload confirms specific binding and activity.
10:35 Coffee Break in the Exhibit Hall with Poster Viewing
PLENARY SESION
11:05 Medical Treatment of HER2 Positive Breast Cancer: Two
Decades of a Fascinating History and More to Come
Martine Piccart, M.D., Ph.D., Head, Medical Oncology, Jules Bordet
Institute; Chair, ESMO (European Society for Medical Oncology)
The talk will cover multiple aspects of anti-HER2 treatment in breast cancer.
It will present a summary of the clinical results obtained with trastuzumab
and several other anti-HER2 drugs in breast cancer (lapatinib, TDM1,
pertuzumab). Issues like the treatment duration, biomarkers of resistance
to treatment will be debated. Finally it will discuss future promising
research strategies: neoadjuvant trials, comparison between anti-HER2
agents, combinations of these drugs and functional imaging.
11:50 Antibody-Drug Conjugates: From Bench to Bedside and Back
Robert Lutz, Ph.D., Vice President, Translational Research & Development,
ImmunoGen, Inc.
Antibody-drug conjugates are emerging as an exciting approach to the
development of antibody-based therapeutics. The growing preclinical and
clinical experience with maytansinoid conjugates such as Kadcyla (T-DM1) is
leading to an enhanced understanding regarding critical attributes for target
antigens, antibodies, payloads and linkers. The translational knowledge
is being incorporated into research and development efforts for the next

generation of ADC candidates.

12:35 End of Cancer Biotherapeutics
http://www.pegsummiteurope.com/PEGS_Europe_Content.aspx?id=123176&libID=123124

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Human Immune System in Health and in Disease |
Tagged Antibody, cancer, Cancer immunotherapy, CD47, EGFR,Genentech, Merrimack Pharmaceuticals, Monoclonal
antibodies | Leave a comment

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